Therapeutic agents for reducing parathyroid hormone levels

ABSTRACT

Compounds having activity for lowering parathyroid hormone levels are described. In one embodiment, the compounds are comprised of a contiguous sequence of subunits, X 1 —X 2 —X 3 —X 4 —X 5 —X 6 —X 7 , wherein the X 1  subunit comprises a thiol-containing moiety and the distribution of charge on the X 2 —X 7  subunits provides the desired activity. Methods of using the compounds for treating hyperparathyroidism, bone disease and/or hypercalcemic disorders are also described, and in particular, methods for lowering plasma PTH and serum calcium are provided. The compounds can be used to treat subjects having, for example: primary, secondary or tertiary hyperparathyroidism; hypercalcemia of malignancy; metastatic bone disease; or osteoporosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.12/846,724, filed Jul. 29, 2010, which claims the benefit of U.S.Provisional Application No. 61/229,695, filed Jul. 29, 2009, and of U.S.Provisional Application No. 61/255,816, filed Oct. 28, 2009, and of U.S.Provisional Application No. 61/313,635, filed Mar. 12, 2010. Each ofthese applications is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

A Sequence Listing is being submitted electronically via EFS in the formof a text file, created Feb. 3, 2012, and named“632008017US03seqlist.txt” (85,400 bytes), the contents of which areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The current subject matter relates to compounds with activity todecrease parathyroid hormone (PTH) levels, to pharmaceuticalcompositions comprising the compounds, and to the use of such compoundsand compositions in methods of treatment, including but not limited totreating hypercalcemia or hyperparathyroidism or modulating in vivo PTHlevels.

BACKGROUND

Calcium homeostasis is the mechanism by which the body maintainsadequate calcium levels. The process is highly regulated, and involves acomplex interplay between calcium absorption, transport, storage inbones, deposition in other tissues, and excretion. PTH is a regulator ofcirculating calcium levels, and functions to increase the concentrationof calcium in the blood by enhancing the release of calcium from bonethrough the process of bone resorption; increasing reabsorption ofcalcium from the renal tubules; and enhancing calcium absorption in theintestine by increasing the production of 1,25-(OH)₂ vitamin D, theactive form of vitamin D. PTH also stimulates phosphorus excretion fromthe kidney, and increases release from bone.

PTH secretion is regulated by the calcium sensing receptor (CaSR), aG-protein coupled receptor expressed by several cell types on thesurface of parathyroid cells, which detects small fluctuations in theconcentration of extracellular calcium ion (Ca²⁺) and responds byaltering the secretion of PTH. Activation of the CaSR by Ca²⁺ inhibitsPTH secretion within seconds to minutes through inhibition of vesiculartransport, and this process may be modulated by protein kinase C(PKC)phosphorylation of the receptor. The CaSR is also expressed onosteoblasts and in the kidney, where it regulates renal Ca²⁺ excretion.

In addition, PTH regulates phosphorus homeostasis. PTH stimulates theparathyroid hormone receptor 1 (PTHR1) on both apical (brush bordermembrane) and basolateral membranes of cells in the GI tract. PTHR1stimulation leads to an increase in urinary excretion of phosphate (Pi)as a consequence of reduction by internalization of the renalNa⁺/phosphate (NaPi-IIa) co-transporter on the brush border membrane.

PTH is also involved in the regulation of osteoblasts and osteoclasts inbone. PTH increases circulating Ca²⁺ by increasing bone resorption andrenal reabsorption of calcium. PTH stimulates osteoblasts to produceRANK ligand (RANKL), which binds to the RANK receptor and activates theosteoclasts, leading to an increase in bone resorption and an increasein serum Ca²⁺. Osteoprotegerin (OPG) is a decoy receptor for RANKL whichblocks bone resorption. Osteoporosis is caused by an imbalance betweenthe processes of bone resorption by osteoclasts and bone formation byosteoblasts.

The human body contains approximately 1 kg of calcium, 99% of whichresides in bone. Under normal conditions, circulating calcium ion (Ca²⁺)is tightly maintained at a level of about 9 to 10 mg/dL (i.e., 2.25-2.5mmol/L; ˜600 mg). Approximately 1 g of elemental calcium (Ca²⁺) isingested daily. Of this amount, approximately 200 mg/day is absorbed,and 800 mg/day is excreted. In addition, approximately 500 mg/day isreleased by bone resorption or is deposited into bone. About 10 g ofCa²⁺ is filtered through the kidney per day, with about 200 mg appearingin the urine, and the remainder being reabsorbed.

Hypercalcemia is an elevated calcium level in the blood. Acutehypercalcemia can result in gastrointestinal (anorexia, nausea,vomiting); renal (polyuria, polydipsia), neuro-muscular (depression,confusion, stupor, coma) and cardiac (bradycardia, first degreeatrio-ventricular) symptoms. Chronic hypercalcemia is also associatedwith gastrointestinal (dyspepsia, constipation, pancreatitis); renal(nephrolithiasis, nephrocalcinosis), neuro-muscular (weakness) andcardiac (hypertension block, digitalis sensitivity) symptoms. Abnormalheart rhythms can result, and EKG findings of a short QT interval and awidened T wave suggest hypercalcemia. Hypercalcemia may be asymptomatic,with symptoms more commonly occurring at high calcium levels (12.0 mg/dLor 3 mmol/l). Severe hypercalcemia (above 15-16 mg/dL or 3.75-4 mmol/l)is considered a medical emergency: at these levels, coma and cardiacarrest can result.

Hypercalcemia is frequently caused by hyperparathyroidism, leading toexcess bone resorption and elevated levels of serum calcium. In primarysporadic hyperparathyroidism, PTH is overproduced by a singleparathyroid adenoma; less commonly, multiple adenomas or diffuseparathyroid gland hyperplasia may be causative. Increased PTH secretionleads to a net increase in bone resorption, with release of Ca²⁺ andphosphate (Pi). PTH also enhances renal reabsorption of Ca²⁺ andinhibits reabsorption of phosphate (Pi), resulting in a net increase inserum calcium and a decrease in phosphate.

Secondary hyperparathyroidism occurs when a decrease in circulatinglevels of Ca²⁺ level stimulates PTH secretion. One cause of secondaryhyperparathyroidism is chronic renal insufficiency (also referred to aschronic kidney disease or CKD), such as that in renal polycystic diseaseor chronic pyelonephritis, or chronic renal failure, such as that inhemodialysis patients (also referred to as end stage renal disease orESRD). Excess PTH may be produced in response to hypocalcemia resultingfrom low calcium intake, GI disorders, renal insufficiency, vitamin Ddeficiency, and renal hypercalciuria. Tertiary hyperparathyroidism mayoccur after a long period of secondary hyperparathyroidism andhypercalcemia.

Malignancy is a common cause of non-PTH mediated hypercalcemia.Hypercalcemia of malignancy, is an uncommon but severe complication ofcancer, affecting between 10% and 20% of cancer patients, and may occurwith both solid tumors and leukemia. The condition has an abrupt onsetand has a very poor prognosis, with a median survival of only six weeks.Growth factors (GF) regulate the production of parathyroidhormone-related protein (PTHrP) in tumor cells. Tumor cells may bestimulated by autocrine GF to increase production of PTHrP, leading toenhanced bone resorption. Tumor cells metastatic to bone may alsosecrete PTHrP, which can resorb bone and release additional GF which inturn act in a paracrine manner to further enhance PTHrP production.

Accordingly, compounds with activity to, for example, modulate PTHlevels and/or calcium levels in vivo are desired.

BRIEF SUMMARY

In one aspect, a compound, comprising the formulaX₁—X₂—X₃—X₄—X₅—X₆—X₇is provided, wherein X₁ is a subunit comprising a thiol-containinggroup; X₅ is a cationic subunit; X₆ is a non-cationic subunit; X₇ is acationic subunit; and at least one, preferably two, of X₂, X₃ and X₄is/are independently a cationic subunit; and wherein the compound hasactivity to decrease parathyroid hormone concentration. In oneembodiment, the decrease in parathyroid hormone concentration is adecrease in blood or plasma parathyroid hormone concentration in asubject treated with the compound relative to the blood or plasmaparathyroid hormone concentration in the subject prior to treatment. Inanother embodiment, the decrease in parathyroid hormone concentration isachieved in the absence of a histamine response.

In another embodiment X₃ and X₄ are non-cationic while X₁, X₅, X₆ and X₇are cationic.

In one embodiment, the X₁ subunit is a thiol-containing amino acidresidue. In another embodiment, the thiol group of the X₁ subunit is anorganic thiol-containing moiety.

In another embodiment, when the X₁ subunit is a thiol-containing aminoacid residue, it is selected from the group consisting of L-cysteine,D-cysteine, glutathione, n-acetylated cysteine, homocysteine andpegylated cysteine.

In yet another embodiment, the organic thiol-containing moiety isselected from thiol-alkyl, or thioacyl moieties such as 3-mercaptopropylor 3-mercaptopropionyl, mercaptopropionic acid, mercaptoacetic acid,thiobenzyl, or thiopropyl. In still another embodiment, theorganic-thiol-containing moiety is mercaptopropionic acid.

In still another embodiment, the X₁ subunit is modified chemically tocomprise an acetyl group, a benzoyl group, a butyl group, or anotheramino acid such as acetylated-beta-alanine.

In yet another embodiment, when the X₁ subunit comprises a thiol moiety,the X₁ subunit is joined by a covalent linkage to a second thiol moiety.

In another embodiment, the formula X₁—X₂—X₃—X₄—X₅—X₆—X₇ is comprised ofa contiguous sequence of amino acid residues (designated herein as(X_(aa1))—(X_(aa2))—(X_(aa3))—(X_(aa4))—(X_(aa5))—(X_(aa6))—(X_(aa7))SEQ ID NO:1) or a sequence of organic compound subunits (non-amino acidresidues).

In another embodiment, the contiguous sequence of amino acid residues isa contiguous sequence of L-amino acid residues, a contiguous sequence ofD-amino acid residues, a contiguous sequence of a mixture of L-aminoacid residues and D-amino acid residues, or a mixture of amino acidresidues and non-natural amino acid residues.

In another embodiment, the contiguous sequence of amino acid residues islinked to a compound to facilitate transport across a cell membrane. Inanother embodiment, the contiguous sequence of amino acid residues islinked to a compound that enhances delivery of the sequence into oracross one or more layers of tissue.

In another embodiment, the contiguous sequence of amino acid residues iscontained within a sequence of amino acid residues from 8-50 amino acidresidues, 8-40 amino acid residues, 8-30 amino acid residues or 8-20amino acid residues in length. In yet another embodiment, the contiguoussequence of amino acid residues is contained within a sequence of aminoacid residues from 8-19 amino acid residues, 8-18 amino acid residues,8-17 amino acid residues, 8-16 amino acid residues, 8-15 amino acidresidues, 8-14 amino acid residues, 8-13 amino acid residues, 8-12 aminoacid residues, 8-11 amino acid residues, 8-10 amino acid residues, or8-9 amino acid residues in length.

In another embodiment, the X₃ subunit is a cationic amino acid residue.

In another embodiment, the X₂ subunit is a non-cationic amino acidresidue, and in another embodiment, the X₄ subunit is a non-cationicamino acid residue. In one embodiment, the non-cationic amino acidresidue is a D-amino acid.

In another embodiment, X₃ and X₄ are cationic D-amino acid residues.

In another embodiment, the X₅ subunit is a D-amino acid residue.

In another aspect, the contiguous sequence in any of the describedcompounds is covalently attached via the thiol-containing group in theX₁ subunit to a second contiguous sequence. For example, the secondcontiguous sequence can be identical to the contiguous sequence (to forma dimer), or can be non-identical, as would be the case when attached toa moiety that facilitates transfer of the contiguous sequence across acell membrane.

In another aspect, a conjugate comprised of the peptide carrrar (SEQ IDNO:2) is provided, where the peptide is conjugated at its N-terminalresidue to a Cys residue.

In one embodiment, the peptide is chemically modified at the N-terminus,the C-terminus, or both.

In another embodiment, the N-terminus of the peptide is chemicallymodified by acetylation and the C-terminus is chemically modified byamidation.

In another embodiment, the conjugate is Ac-c(C)arrrar-NH₂ (SEQ ID NO:3).

In another aspect, a method of treating secondary hyperparathyroidism(SHPT) in a subject is contemplated, wherein a compound as describedherein is provided to the subject. In various embodiments, the subjectcan be suffering from chronic kidney disease or other condition.

In another aspect, a method of decreasing parathyroid hormone in asubject is contemplated, wherein a compound as described herein isprovided to the subject.

In another aspect, a treatment regimen is provided, the regimencomprising providing a compound according to any of those describedherein, in combination with a second agent.

In one embodiment, the second therapeutic agent is vitamin D, a vitaminD analog or cinacalcet hydrochloride.

In any of the aspects or embodiments described herein, any one or moreof the sequences is contemplated to be individually excepted or removedfrom the scope of the claims. In certain embodiments, the peptidesidentified by any one or more of SEQ ID NOs: 162-182, individually or inany combination, are excluded from the claimed compounds, compositionsand methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of parathyroid hormone level, as percent of thebaseline pre-dose value, as a function of time, in hours, in rats withacute renal insufficiency (1K1C model), where the rats were dosed withAc-crrrr-NH₂ (SEQ ID NO:4, diamonds), Ac-crrrrr-NH₂ (SEQ ID NO:5, closedsquares), Ac-crrrrrr-NH₂ (SEQ ID NO:6, triangles), Ac-crrrrrrr-NH₂ (SEQID NO:7, open squares), or saline control (x symbols);

FIG. 2A is a graph of IP₁ concentration, in nM, as a function ofcompound concentration of Ac-carrrar-NH₂ (SEQ ID NO:26, squares) andAc-arrrar-NH₂ (SEQ ID NO:29, triangles), as a measure of the compound'sability to activate the human CaSR in an in vitro cell assay when thehuman CaSR is expressed as a stable transfected HEK-293 cell line;

FIG. 2B shows the reduction in PTH concentration upon in vivoadministration of peptides identified as SEQ ID NO:26 (Ac-carrrar-NH₂)(squares) and as SEQ ID NO:29 (Ac-arrrar-NH₂) (diamonds), where thepeptides were administered as an IV bolus to normal Sprague Dawley ratsat doses of 9 mg/kg for SEQ ID NO:29 and at 0.5 mg/kg for SEQ ID NO:26.An intravenous (IV) bolus of saline was used as a control (dashed line).Plasma PTH levels were assessed prior to dosing and 1, 2, 3 and 4 hoursafter dosing. Results are presented as group average±standard deviation(SD), and PTH is shown as percent of the baseline pre-dose value;

FIG. 3 is a bar graph that compares the release of histamine followingIV bolus administration of various compounds in normal Sprague Dawleyrats, where the compounds Ac-crrrr-NH₂ (SEQ ID NO:4), Ac-crrrrr-NH₂ (SEQID NO:5), Ac-crrrrrr-NH₂ (SEQ ID NO:6) and Ac-crrrrrrrr-NH₂ (SEQ IDNO:41) were dosed in an equimolar IV bolus dose of 2.1 μmol/kg, andplasma histamine was measured before dosing (pre-dose), 5, 15 and 30minutes after dosing;

FIG. 4 is a bar graph that compares the release of histamine followingIV bolus administration of two compounds in normal Sprague Dawley rats,where the compounds Ac-c(C)arrrar-NH₂ (SEQ ID NO:3, cross hatched bars)and Ac-crrrrrr-NH₂ (SEQ ID NO:6, open bars) were dosed at 3 mg/kg, andplasma histamine was measured before dosing (time zero) and 5, 15 and 30minutes after dosing;

FIG. 5 is a graph of parathyroid hormone level, as percent of thebaseline pre-dose value, as a function of time, in hours, in normal ratsdosed with 0.5 mg/kg by IV bolus of Ac-crrrrrr-NH₂ (SEQ ID NO:6,diamonds), Ac-carrrrr-NH₂ (SEQ ID NO:8, squares), Ac-crarrrr-NH₂ (SEQ IDNO:9, triangles), Ac-crrarrr-NH₂ (SEQ ID NO:10, x symbols),Ac-crrrarr-NH₂ (SEQ ID NO:11, * symbols), Ac-crrrrar-NH₂ (SEQ ID NO:12,circles) or Ac-crrrrra-NH₂ (SEQ ID NO:13, +symbols);

FIGS. 6A-6B are graphs of parathyroid hormone level, as percent of thebaseline pre-dose value, as a function of time, in hours, in healthyrats dosed with 0.5 mg/kg by IV bolus of Ac-carrrar-NH₂ (SEQ ID NO:26,open diamonds), Ac-crrarar-NH₂ (SEQ ID NO:25, open squares),Ac-caarrrr-NH₂ (SEQ ID NO:22, triangles), Ac-crraarr-NH₂ (SEQ ID NO:17,closed squares), Ac-c(C)arrrar-NH₂ (SEQ ID NO:3, diamonds FIG. 6B),Ac-craarrr-NH₂ (SEQ ID NO:24, x symbols in FIG. 6A); Ac-c(C)rrarar-NH₂(SEQ ID NO:28, x symbols, FIG. 6B);

FIG. 7 shows the decrease in parathyroid hormone levels in the blood asa function of time, for the compound Ac-c(C)arrrar-NH₂ (SEQ ID NO:3)administered as an IV bolus to normal Sprague Dawley rats at doses of 1mg/kg (diamonds), 0.5 mg/kg (squares), 0.3 mg/kg (triangles), and 0.1mg/kg (x symbols). An intravenous (IV) bolus of saline (circles) wasused as a control. Plasma PTH levels were assessed prior to dosing andat 1, 2, 3 and 4 hours after dosing.

FIG. 8 is a graph of parathyroid hormone level, as percent of thebaseline pre-dose value, as a function of time, in hours, in rats withacute renal insufficiency (1K1C model), in rats with 1K1C model of acuterenal insufficiency, where the rats were dosed via IV bolus with thecompound Ac-c(C)arrrar-NH₂ (SEQ ID NO:3) at doses of 3 mg/kg (diamonds),1 mg/kg (triangles), 0.5 mg/kg (squares) and 0.3 mg/kg (x symbols), orsaline (squares); the dashed line in FIG. 8 indicating baseline PTHlevel pre-dosing;

FIG. 9 is a graph of parathyroid hormone level, as percent of thebaseline pre-dose value, as a function of time, in hours, in rats dosedintravenously with saline (x symbols) or with the compoundsAc-crrrrrr-NH₂ (SEQ ID NO:6, open diamonds), and Ac-carrrar-NH₂ (SEQ IDNO:26, open squares) at 1 mg/kg via a 30-minute IV infusion, whereplasma PTH levels were assessed prior to dosing, 16 hours and 24 hoursafter dosing;

FIG. 10 is a graph of parathyroid hormone level, as percent of thebaseline pre-dose value, as a function of time, in hours, in rats withacute renal insufficiency (1K1C model), where the rats were dosed via IVbolus with the compounds Ac-c(C)arrrar-NH₂ (SEQ ID NO:3, squares, *symbols) and Ac-c(Ac-C)arrrar-NH₂ (SEQ ID NO:146, triangles, diamonds)at doses of 0.3 mg/kg (squares, triangles) and 0.5 mg/kg (*, diamonds);

FIG. 11 is a graph of parathyroid hormone level, as percent of thebaseline pre-dose value, as a function of time, in hours, in ratstreated via micropore-facilitated transdermal delivery of Ac-crrrrrr-NH₂(SEQ ID NO:6, two animals, squares and triangles) or with saline viatransdermal delivery (diamonds);

FIG. 12 is a graph of parathyroid hormone level, as percent of thebaseline pre-dose value, as a function of time, in hours, in ratstreated via micropore-facilitated transdermal delivery ofAc-c(C)arrrar-NH₂ (SEQ ID NO:3);

FIG. 13 is a graph of mean PTH (as percent of baseline) during andfollowing a 6 hour IV infusion of Ac-c(C)arrrar-NH₂ (SEQ ID NO:3) innormal Sprague-Dawley rats, where the compound was infused at rates of 1μg/kg/hr (squares), 3 μg/kg/hr (circles), and 10 μg/kg/hr (triangles);

FIG. 14A shows PTH (as a percent of baseline) during and following a 6hour IV infusion of Ac-c(C)arrrar-NH₂ (SEQ ID NO:3) in the 1K1C ratmodel of acute renal insufficiency, where rats were intravenouslyinfused at dose rates of 30 μg/kg/hr (diamonds) and 100 μg/kg/hr(squares);

FIG. 14B is a bar graph showing serum calcium, in mg/dL, for the 1K1Cmodel rats treated as in FIG. 14A.

The present subject matter may be understood more readily by referenceto the following detailed description of the preferred embodiments andthe examples included herein.

DETAILED DESCRIPTION I. Definitions

Within this application, unless otherwise stated, definitions of theterms and illustration of the techniques of this application may befound in any of several well-known references such as: Sambrook, J., etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press (1989); Goeddel, D., ed., Gene Expression Technology,Methods in Enzymology, 185, Academic Press, San Diego, Calif. (1991);“Guide to Protein Purification” in Deutshcer, M. P., ed., Methods inEnzymology, Academic Press, San Diego, Calif. (1989); Innis, et al., PCRProtocols: A Guide to Methods and Applications, Academic Press, SanDiego, Calif. (1990); Freshney, R. I., Culture of Animal Cells: A Manualof Basic Technique, 2nd Ed., Alan Liss, Inc. New York, N.Y. (1987);Murray, E. J., ed., Gene Transfer and Expression Protocols, pp. 109-128,The Humana Press Inc., Clifton, N.J. and Lewin, B., Genes VI, OxfordUniversity Press, New York (1997).

As used herein, the singular form “a”, “an”, and “the” include pluralreferences unless indicated otherwise. For example, “a” modulatorpeptide includes one of more modulator peptides.

As used herein a compound has “activity to decrease parathyroid hormonelevel” or “PTH-lowering activity” when the compound, upon administrationto a subject, lowers plasma parathyroid hormone (PTH) relative to theplasma PTH concentration prior to administration of the compound. In oneembodiment, the decrease in PTH level is at least 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or 95% lower one hour after compound administrationthat the PTH level prior to administration of the compound.

As used herein, “absence of a histamine response” or “lack of ahistamine response” intends a dose of a compound that produces a lessthan 15-fold, 14-fold, 13-fold, 12-fold, 11-fold, 10-fold, 9-fold,8-fold, 7-fold, 6-fold, 5-fold, 4-fold, or 3-fold increase in histamine,measured in vitro in an assay as described herein, where the fold changeis determined based on histamine levels before incubation with thecompound and after 15 minutes incubation with compound.

As used herein, “amino acid” refers to natural and non-natural aminoacids. The twenty naturally occurring amino acids (L-isomers) aredesignated by the three letter code with the prefix “L-” (except forglycine which is achiral) or by the one letter code in upper-case:alanine (“L-Ala” or “A”), arginine (“L-Arg” or “R”), asparagine (“L-Asn”or “N”), aspartic acid (“L-Asp” or “D”), cysteine (“L-Cys” or “C”),glutamine (“L-Gln” or “Q”), glutamic acid (“L-Glu” or “E”), glycine(“Gly” or “G”), histidine (“L-His” or “H”), isoleucine (“L-Ile” or “I”),leucine (“L-Leu” or “L”), lysine (“L-Lys” or “K”), methionine (“L-Met”or “M”), phenylalanine (“L-Phe” or “F”), proline (“L-Pro” or “P”),serine (“L-Ser” or “S”), threonine (“L-Thr” or “T”), tryptophan (“L-Trp”or “W”), tyrosine (“L-Tyr” or “Y”) and valine (“L-Val” or “V”).L-norleucine and L-norvaline may be represented as (NLeu) and (NVal),respectively. The nineteen naturally occurring amino acids that arechiral have a corresponding D-isomer which is designated by the threeletter code with the prefix “D-” or by the lower-case one letter code:alanine (“D-Ala” or “a”), arginine (“D-Arg” or “r”), asparagine (“D-Asn”or “a”), aspartic acid (“D-Asp” or “d”), cysteine (“D-Cys” or “c”),glutamine (“D-Gln” or “q”), glutamic acid (“D-Glu” or “e”), histidine(“D-His” or “h”), isoleucine (“D-Ile” or “i”), leucine (“D-Leu” or “l”),lysine (“D-Lys” or “k”), methionine (“D-Met” or “m”), phenylalanine(“D-Phe” or “f”), proline (“D-Pro” or “p”), serine (“D-Ser” or “s”),threonine (“D-Thr” or “t”), tryptophan (“D-Trp” or “w”), tyrosine(“D-Tyr” or “y”) and valine (“D-Val” or “v”). D-norleucine andD-norvaline may be represented as (dNLeu) and (dNVal), respectively.Although “amino acid residue” is often used in reference to a monomericsubunit of a peptide, polypeptide or protein, and “amino acid” is oftenused in reference to a free molecule, usage of these terms in the artoverlaps and varies. The term “amino acid” and “amino acid residue” areused interchangeably and may refer to a free molecule or a monomericsubunit of a peptide, polypeptide or protein, depending on context.

To determine the percent “homology” or percent “identity” of two aminoacid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of onepolypeptide for optimal alignment with the other polypeptide). The aminoacid residues at corresponding amino acid positions are then compared.When a position in one sequence is occupied by the same amino acidresidue as the corresponding position in the other sequence, then themolecules are identical at that position. As used herein amino acid ornucleic acid “homology” is equivalent to amino acid or nucleic acid“identity”. Accordingly, the percent sequence identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., percent sequence identity=numbers of identicalpositions/total numbers of positions×100). Percent sequence identitybetween two polypeptide sequences can be determined using the Vector NTIsoftware package (Invitrogen Corporation, 5791 Van Allen Way, Carlsbad,Calif. 92008). A gap opening penalty of 10 and a gap extension penaltyof 0.1 are used for determining the percent identity of twopolypeptides. All other parameters are set at the default settings.

A “cationic amino acid” intends an amino acid residue that has a netpositive charge at physiologic pH (7.4), as is the case, for example, inthe amino acid residues where the side chain, or “R group”, contains anamine functional group or other functional group that can accept aproton to become positively charged at physiologic pH, such as aguanidine or imidazole moiety. Cationic amino acid residues includearginine, lysine, histidine, 2,3-diaminopropionic acid (Dap),2,4-diaminobutyric acid (Dab), ornithine, and homoarginine.

A “cationic subunit” intends a subunit that has a net positive charge atphysiologic pH (7.4).

As used herein, “conservative amino acid substitutions” aresubstitutions which do not result in a significant change in theactivity or tertiary structure of a selected polypeptide or protein.Such substitutions typically involve replacing a selected amino acidresidue with a different amino acid residue having similarphysico-chemical properties. Groupings of amino acids and amino acidresidues by physico-chemical properties are known to those of skill inthe art. For example, among the naturally-occurring amino acids,families of amino acid residues having similar side chains have beendefined in the art, and include basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

As used herein, “chemical cross-linking” refers to covalent bonding oftwo or more molecules.

A peptide or peptide fragment is “derived from” a parent peptide orpolypeptide if it has an amino acid sequence that is identical orhomologous to at least a contiguous sequence of five amino acidresidues, more preferably eight amino acid residues, of the parentpeptide or polypeptide. The compounds described herein may be in theform of pharmaceutically acceptable salts. Pharmaceutically acceptablesalts include acid addition salts, such as hydrochloride, hydrobromide,sulfurate, nitrate, phosphorate, acetate, propionate, glycolate,pyruvate, oxalate, malate, malonate, succinate, maleate, fumarate,tartarate, citrate, benzoate, cinnamate, mandelate, methanesulfonate,ethanesulfonate, p-toluene-sulfonate, salicylate and the like, and baseaddition salts, such as sodium, potassium, calcium, magnesium, lithium,aluminum, zinc, ammonium, ethylenediamine, arginine, piperazine and thelike.

As used herein, the term “hyperparathyroidism” refers to primary,secondary and tertiary hyperparathyroidism, unless otherwise indicated.

The term “intradermal” intends that in the methods of treatmentdescribed herein a therapeutically effective amount of a calcimimeticcompound is applied to skin to deliver the compound to layers of skinbeneath the stratum corneum, and thus achieve a desired therapeuticeffect.

As used herein, an “isolated” or “purified” polypeptide or biologicallyactive portion thereof is free of some of the cellular material whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. The language “substantially freeof cellular material” includes preparations of polypeptides in which thepolypeptide is separated from some of the cellular components of thecells in which it is naturally or recombinantly produced. When thepolypeptide or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume of the polypeptide preparation. The language “substantially freeof chemical precursors or other chemicals” includes preparations ofpolypeptides in which the polypeptide is separated from chemicalprecursors or other chemicals that are involved in the synthesis of thepolypeptide. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of apolypeptide having less than about 30% (by dry weight) of chemicalprecursors or other chemicals, preferably less than about 20% chemicalprecursors or other chemicals, more preferably less than about 15%chemical precursors or other chemicals, still more preferably less thanabout 10% chemical precursors or other chemicals, and most preferablyless than about 5% chemical precursors or other chemicals. In preferredembodiments, isolated polypeptides, or biologically active portionsthereof, lack contaminating polypeptides from the same organism fromwhich the domain polypeptide is derived.

As used herein, “macromolecule” refers to a molecule, such as a peptide,polypeptide, protein or nucleic acid, that typically has a molecularweight greater than about 900 Daltons.

A “non-cationic amino acid” intends an amino acid residue that has nocharge or a net negative charge at physiologic pH (7.4), as is the case,for example, in the amino acid residues where the side chain, or “Rgroup”, is neutral (neutral polar and neutral non-polar) and acidic.Non-cationic amino acids include those residues with an R group that isa hydrocarbon alkyl or aromatic moiety (e.g., valine, alanine, leucine,isoleucine, phenylalanine); a neutral, polar R group (asparagine,cysteine, glutamine, serine, threonine, tryptophan, tyrosine); or aneutral, non-polar R group (glycine, methionine, proline, valine,isoleucine). Non-cationic amino acids with an acidic R group includeasparatic acid and glutamic acid.

A “polymer” refers to a linear chain of two or more identical ornon-identical subunits joined by covalent bonds.

As used herein, “peptide” and “polypeptide” refer to any polymer made upof a chain of amino acid residues linked by peptide bonds, regardless ofits size. Although “protein” is often used in reference to relativelylarge polypeptides, and “peptide” is often used in reference to smallpolypeptides, usage of these terms in the art overlaps and varies. Thus,for simplicity, the term “peptide” will be used herein, although in somecases the art may refer to the same polymer as a “polypeptide.” Unlessotherwise indicated, the sequence for a peptide is given in the orderfrom the amino terminus to the carboxyl terminus.

A “thiol-containing group” or “thiol-containing moiety” as used hereinintends a functional group comprising a sulfur-hydrogen bond (—SH), andthat is capable of reacting with another thiol under physiologicconditions to form a disulfide bond. A thiol that is capable of forminga disulfide bond with another thiol is referred to herein as a “reactivethiol.” In a preferred embodiment the thiol-containing group is lessthan 6 atoms away from the backbone of the compound. In a more preferredembodiment, the thiol-containing group has the structure(—SH—CH₂—CH₂—C(O)—O—)—.

As used herein, “small molecule” refers to a molecule other than amacromolecule, such as an organic molecule, and typically has amolecular weight of less than 1000 daltons.

As used herein, “subject” refers to a human subject or an animalsubject.

A “subunit” intends a monomeric unit that is joined to more than oneother monomeric unit to form a polymeric compound, where a subunit isthe shortest repeating pattern of elements in the polymeric compound.Exemplary subunits are amino acids, which when linked form a polymercompound such as those referred to in the art as a peptide, apolypeptide or a protein.

As used herein, a “therapeutically effective amount” is an amountrequired to produce a desired therapeutic effect. For example, inmethods for reducing serum calcium in hypercalcemic subjects, atherapeutically effective amount is the amount required to reduce serumcalcium levels by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or 25%. Calcium may bemeasured as total calcium or as ionized calcium. By way of anotherexample, in methods for lowering in vivo PTH, a therapeuticallyeffective amount is the amount required to reduce PTH levels by at least1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20% or 25%.

As used herein, the term “transdermal” means that in the methods oftreatment described herein a therapeutically effective amount of acalcimimetic agent is applied to skin to deliver the compound tosystemic circulation and thus achieve a desired therapeutic effect.

Unless otherwise specified, all documents referred to herein areincorporated by reference in their entirety.

II. Compounds

In one aspect, a compound comprising the sequence of subunitsX₁—X₂—X₃—X₄—X₅—X₆—X₇ is provided, where X₁ is a subunit comprising athiol group; X₅ is a cationic subunit; X₆ is a non-cationic subunit; X₇is a cationic a subunit; and at least two of X₂, X₃ and X₄ areindependently a cationic subunit. The compounds have activity todecrease parathyroid hormone (PTH) levels and/or decrease calcium levelsin a subject's blood. A decrease in parathyroid hormone levels, as willbe illustrated below, intends a lowering of plasma or blood PTHconcentration in a subject relative to the plasma or blood PTHconcentration prior to treatment with the compound. In one embodiment,the compound achieves a reduction in plasma PTH concentration by atleast 50% within one hour after dosing, relative to the plasma PTH priorto dosing. The compounds are exemplified by peptides, although a skilledartisan will appreciate that non-peptidic compounds that have thedesired activity can be designed based on the structure-activityrelationship studies described herein.

As used herein parathyroid hormone or PTH is an 84 amino acid peptideproduced by the parathyroid gland and its breakdown products. Besidesfull length PTH (which consists of residues 1-84 and is sometimesreferred to as “intact” of “bioactive” PTH) various PTH fragmentsgenerated by proteolysis and other routes of metabolism are present inblood. The amino-terminal 1-34 region of the intact PTH molecule isbiologically active. This region of the molecule contains the amino acidsequence that enables PTH to bind to the parathyroid hormone receptorsin target tissues. The middle and carboxy-terminal 35-84 region of theintact PTH molecule is believed to be biologically inert but possessesimmunological reactivity. PTH 7-84 is thought to exert effects that areopposite to those of 1-84 PTH. Various assays have been developed tomeasure PTH levels including various breakdown products and are reviewedby Souberbielle et. al., Kidney International, 77:93-100 (2010), whichis incorporated herein by reference. In one embodiment, a compoundhaving activity to decrease PTH level as defined herein is ascertainedusing a validated PTH quantification method that detects the intactbioactive form of PTH (1-84), and commercially available kits are knownin the art (e.g., see Example 3 herein).

In a first study, compounds containing 4 to 7 cationic (e.g., arginine)subunits were generated and tested for their ability to lower PTH ascompared with baseline PTH values and saline-treated animals.Specifically, a 1K1C model of acute renal insufficiency was establishedfor use in characterizing the PTH-lowering activity in a renaldysfunction environment. The 1K1C model is described in Example 1A, andthe compounds synthesized for testing included (i) Ac-crrrr-NH₂ (SEQ IDNO:4), (ii) Ac-crrrrr-NH₂ (SEQ ID NO:5), (iii) Ac-crrrrrr-NH₂(SEQ IDNO:6), (iv) Ac-crrrrrrr-NH₂ (SEQ ID NO:7) and (v) saline control.

As described in Example 1B, the compounds identified as SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6 and SEQ ID NO:7 were each administered by a30-minute IV infusion to 1K1C model animals. FIG. 1 shows the reductionin plasma PTH levels as a percent of the pre-dosing (baseline) level.All four compounds dosed at 3 mg/kg produced a significant drop inplasma PTH, but differences in the potency and duration of PTH reductionsuggest a relationship between the net positive charge and PTH-loweringactivity. For example, the compound Ac-crrrrrr-NH₂ (SEQ ID NO:6;triangles) with six cationic (arginine) subunits had increased efficacyas well as the duration of action compared to the compounds Ac-crrrr-NH₂(SEQ ID NO:4; diamonds) and Ac-crrrrr-NH₂ (SEQ ID NO:5; squares),containing four and five cationic (arginine) subunits, respectively.Surprisingly, the compound Ac-crrrrrr-NH₂ (SEQ ID NO:6; triangles) withsix cationic (arginine) subunits had increased duration of actioncompared to the compound Ac-crrrrrrr-NH₂ (SEQ ID NO:7, open squares)with seven cationic (arginine) residues, suggesting that activity orpotency of the compounds does not correlate merely with increasingcationic charge of the compound. That is, the compound Ac-crrrrrrr-NH₂(SEQ ID NO:7) with seven cationic subunits (arginine residues) produceda similar initial drop in PTH as the compounds with fewer cationicresidues, but over the 24 hours following dosing was less efficaciousthan Ac-crrrrrr-NH₂ (SEQ ID NO:6) and Ac-crrrrr-NH₂ (SEQ ID NO:5). Theselatter two compounds produced a mean PTH reduction of ˜40% and 60% atthe 24 hour time point, respectively. Both the extent of PTH reductionand duration of PTH are important criteria for obtaining optimaltherapeutic benefit for patients in need of treatment. It should benoted that the compounds in this study were administered at the samemg/kg dose but, due to differences in molecular weight, a differentnumber of moles of each compound was actually dosed. Therefore,Ac-crrrrrr-NH₂ (SEQ ID NO:6) was significantly more potent thanAc-crrrr-NH₂ (SEQ ID NO:4) and Ac-crrrrr-NH₂ (SEQ ID NO:5) on a per molebasis.

Further studies were done to explore the structure-activity relationshipof the compounds. The compound Ac-crrrrrr-NH₂ (SEQ ID NO:6) was modifiedby sequential replacement of an arginine residue with an alanine residueat each of the subunit positions X₂—X₇. The compounds were characterizedin an in vitro human calcium-sensing receptor (CaSR) assay, described inExample 2, wherein HEK 293 cells that express the human calcium-sensingreceptor were used to measure activity of exemplary compounds. Withoutwishing to be bound by theory, it is thought that the mechanism by whichthe described compounds lower PTH in vivo is through the activation ofthe CaSR, which is expressed in the parathyroid gland and controls PTHsecretion. Activation of the CaSR leads to an increase in intracellularcalcium and inositol-3-phosphate (IP3) and the subsequent accumulationof inositol-phosphate-1 (IP₁). Accordingly, in this in vitro assay, thehalf maximal effective concentration of compound to reduce IP₁generation by 50% was determined (EC₅₀). The same compounds were alsotested in vivo to determine their PTH-lowering activity, as described inExample 3. Results are shown in Table 1. The numbers in the columntitled “% PTH AUC (1-4 hrs) of saline control” of Table 1 defineactivity as reduction in Area Under the Curve (AUC) of PTH over 4 hoursas a percent of PTH AUC derived from saline-treated control rats. Forexample, an AUC (compound treated)/AUC (saline control)*100 that isequal to 0 would be indicative of a highly active PTH-lowering compoundthat completely suppresses PTH (to an undetectable level) for 4 hoursafter a single IV administration of isoflurane (IF)-anesthetized normalrats. In contrast, a value of AUC (compound treated)/AUC (salinecontrol)*100 that is equal to or greater than 100 would be indicative ofan inactive compound.

TABLE 1 In vitro and In vivo activity of Exemplary Compounds In vivoactivity in normal rats** % PTH reduction of baseline at 1 0.5 mg/kghour post IV IV bolus % admin. of PTH AUC In vitro 0.5 mg/kg of salineEC₅₀ SEQ ID NO. Structure* compound control (uM) SEQ ID NO: 6Ac-crrrrrr-NH₂ 4 0 0.5 SEQ ID NO: 8 Ac-carrrrr-NH₂ 0 0 1.1 SEQ ID NO: 9Ac-crarrrr-NH₂ 0 7 1.0 SEQ ID NO: 10 Ac-crrarrr-NH₂ 0 0 1.1 SEQ ID NO:11 Ac-crrrarr-NH₂ 9 45 5.9 SEQ ID NO: 12 Ac-crrrrar-NH₂ 3 3 0.45 SEQ IDNO: 13 Ac-crrrrra-NH₂ 4 28 1.1 Saline 128 100 ND *Bolded fond indicatesD-alanine substitutions of cationic amino acids (D-arginine in SEQ IDNO: 6. **PTH reduction following 0.5 mg/kg IV administration inisofluorane-anesthetized normal rats - PTH was measured at 1, 2, 3 and 4hours post administration and cumulative AUC was calculated. PTH datawere calculated according to the following formula:AUC_(cmpd treated)/AUC_(saline control) * 100.

In Table 1, the compounds Ac-crrrrrr-NH₂ (SEQ ID NO:6), Ac-carrrrr-NH₂(SEQ ID NO:8) and Ac-crrarrr-NH₂ (SEQ ID NO:10) were quite potent, asevidenced by the decrease in percent PTH to below the detection limit oressentially zero as measured in vivo after a single IV aministration innormal rats. Substitution of the cationic (arginine) residue atpositions 2, 3, 4 or 7 of Ac-crrrrrr-NH₂ (SEQ ID NO:6) resulted in anapproximately two-fold loss in in vitro potency. The substitution atposition 5 to produce the compound Ac-crrrarr-NH₂ (SEQ ID NO:11)produced a 5-10 fold reduction in in vitro potency, although the in vivopercent PTH AUC reduction of 45% could be sufficiently active forclinical therapy. Surprisingly, the substitution of the cationicarginine residue at position 6 with the uncharged (alanine) residueactually improved potency. The data illustrate that cationic anduncharged residues at different positions are not all equal and thereare changes in activity as a result of change in the compound structure.

To further evaluate the effect of change in activity as a function ofchange in compound structure, another series of analogs ofAc-crrrrrr-NH₂ (SEQ ID NO:6) was generated containing double amino acidsubstitutions, where two cationic (arginine) residues were replaced byuncharged (alanine) residues, and tested for potency. Data are shown inTable 2. It is worth noting that this series of compounds have the samenet cationic charge as SEQ ID NO:4 (four cationic residues) yetsurprisingly some are very active (SEQ ID NO:26) with very low % PTH AUCof saline control while others are inactive (e.g., SEQ ID NO:14).Unexpectedly, this suggests that position of charges as well as totalcationic charge can influence potency of the compounds for reduction ofPTH. The data shown in Table 2 is consistent with the data shown inTable 1 suggesting that the cationic residues of SEQ ID NO:6 areessential at positions 5 and 7 but is not required at position 6, forPTH-lowering activity.

TABLE 2 In vivo Activity of Exemplary Compounds In vivo activity innormal rats** % PTH reduction of baseline at 1 0.5 mg/kg hour post IV IVbolus administration % PTH AUC Compound of 0.5 mg/kg of saline SEQ IDNO. Structure* compound control* Saline Saline 128 100 SEQ ID NO: 14Ac-crrarra-NH₂ 86 130 SEQ ID NO: 15 Ac-cararrr-NH₂ 75 116 SEQ ID NO: 16Ac-carrarr-NH₂ 118 105 SEQ ID NO: 17 Ac-crraarr-NH₂ 39 102 SEQ ID NO: 18Ac-crararr-NH₂ 72 87 SEQ ID NO: 19 Ac-carrrra-NH₂ 29 72 SEQ ID NO: 20Ac-crarrra-NH₂ 45 69 SEQ ID NO: 21 Ac-crrraar-NH₂ 36 50 SEQ ID NO: 22Ac-caarrrr-NH₂ 24 48 SEQ ID NO: 23 Ac-crarrar-NH₂ 0 43 SEQ ID NO: 24Ac-craarrr-NH₂ 8 9 SEQ ID NO: 25 Ac-crrarar-NH₂ 4 6 SEQ ID NO: 26Ac-carrrar-NH₂ 0 1 SEQ ID NO: 27 Ac-c(C)arrrar-NH₂ 2 8 SEQ ID NO: 28Ac-c(C)rrarar-NH₂ 0 16 *Bolded font indicates respsective D-alaninesubstitutions of cationic amino acids (D-arginine) in Ac-crrrrrr-NH₂(SEQ ID NO: 6) **PTH reduction following 0.5 mg/kg IV administration inisofluorine-anesthetized normal rats - PTH was measured at 1, 2, 3 and 4hours post administration and cumulative AUC was calculated. PTH datawere calculated according to the following formula:AUC_(cmpd treated)/AUC_(saline control) * 100.

The data in Table 2 illustrates the structural changes that influenceactivity. In one embodiment, the compound is Ac-caarrrr-NH₂ (SEQ IDNO:22) and in another embodiment, the compound is Ac-craarrr-NH₂ (SEQ IDNO:24).

Further structure-activity relationship studies were conducted using thein vitro cell assay in HEK 293 cells that express the humancalcium-sensing receptor, as described in Example 4. The ability of thepeptides Ac-carrrar-NH₂ (SEQ ID NO:26) and Ac-arrrar-NH₂ (SEQ ID NO:29)to activate the human CaSR was ascertained by the measuring accumulationof inositol monophosphate (IP₁), which is reflective of IP₃ production.IP₃ production is an important cell signaling second messenger and itsproduction is a direct downstream consequence of CaSR activation.Accumulation of IP₁ following IP₃ production can be obtained by treatingthe cells used in the assay with Lithium Chloride (LiCl₂) which inhibitsthe enzyme that converts IR, to inositol. In the studies described inExample 4 accumulation of IP₁ was measured in the presence of theexemplary compounds Ac-carrrar-NH₂ (SEQ ID NO:26) and Ac-arrrar-NH₂ (SEQID NO:29). Results are shown in FIG. 2A.

The concentration of IP₁ is reported as nM along the Y-axis and compoundconcentrations of SEQ ID NO:26 or SEQ ID NO:29 are reported as M alongthe X-axis. Absence of the N-terminal D-cysteine residue from SEQ IDNO:29 dramatically reduced the ability of the compound to activate theCaSR as compared to SEQ ID NO:26. That is, elimination of the N-terminalcysteine residue significantly reduced the potency of the compound, asthe peptides Ac-carrrar-NH (SEQ ID NO:26) and Ac-arrrar-NH₂ (SEQ IDNO:29) differ only by the presence or absence of the N-terminalD-cysteine.

The contribution of the thiol-containing group in the X₁ subunit of thecompound (e.g., in certain embodiments where the compound is a peptideon the N-terminal residue), was also investigated in an in vivo study.The PTH-lowering activity of the peptides identified as SEQ ID NO:26(Ac-carrrar-NH₂) and as SEQ ID NO:29 (Ac-arrrar-NH₂) was evaluated invivo according to the procedures in Example 4. Plasma PTH levels wereassessed prior to dosing and at 1, 2, 3 and 4 hours after dosing. Theresults are shown in FIG. 2B. As seen, a 0.5 mg/kg dose of the peptideAc-carrrar-NH₂ (SEQ ID NO:26) (squares) decreased PTH bloodconcentration to a non-detectable level for up to 4 hours after dosing.In contrast, the peptide lacking an N-terminal residue with athiol-containing group, Ac-arrrar-NH₂ (SEQ ID NO:29), diamonds, did notreduce PTH concentration, even at a substantially higher dose (i.e., 9mg/kg).

The structure-activity relationship of the thiol-containing group in theX₁ subunit of the compound was further analyzed by preparing compoundswith differing X₁ subunits. The compounds, shown in Table 3, were testedin vivo in normal rats for activity to reduce PTH.

TABLE 3 In vivo Activity of Exemplary Compounds In vivo activity innormal rats* 0.5 mg/kg IV bolus % PTH AUC of SEQ ID NO. CompoundStructure saline control** Saline Saline 100 SEQ ID NO: 6 Ac-crrrrrr-NH₂3 SEQ ID NO: 30 Ac-bAla-crrrrrr-NH₂ 0 SEQ ID NO: 31 Mpa-rrrrrr-NH₂ 2 SEQID NO: 32 Ac-dHcy-rrrrrr-NH₂ 21 SEQ ID NO: 33 Ac-dPen-rrrrrr-NH₂ 9*Bolded font indicates respective substitution of thiol-containingresidue (D-cysteine) in Ac-crrrrrr-NH₂ (SEQ ID NO: 6). **PTH reductionfollowing 0.5 mg/kg IV administration in isoflurane-anesthetized normalrats - PTH was measured at 1, 2, 3 and 4 hours post administration andcumulative AUC was calculated. PTH data were calculated according to thefollowing formula: AUC_(cmpd treated)/AUC_(saline control) * 100.

The data in Table 3 illustrates that the thiol-containing X₁ subunit canbe varied. Compounds with the following in the N-terminal residue weretested—D-cysteine (cys), D-penicillamine (dPen), d-homocysteine (dHcy)and mercaptopropionic acid (Mpa). In addition, a natural or non-naturalamino acid, such as beta alanine, can be conjugated to the N-terminalthiol-containing residue. The data illustrates that cationic compoundssuch as Ac-crrrrrr-NH₂ (SEQ ID NO:6) containing differentthiol-containing groups in the X₁ subunit effectively reduce PTH invivo. Substituting the N-terminal cysteine residue with methionine,which does not contain a thiol group, resulted in a compound with verypoor in vivo PTH-lowering activity (data not shown).

Based on the studies above, compounds of the contiguous sequence ofsubunits X₁—X₂—X₃—X₄—X₅—X₆—X₇, where X₁ is a subunit comprising athiol-containing group, have activity to decrease parathyroid hormonelevels. In one embodiment, the thiol-containing group on the X₁ subunitis selected from the group consisting of thiol-containing amino acidresidues and organic thiol-containing moieties. In another embodiment,the thiol-containing group is capable of reacting with another thiolgroup under physiologic pH and temperature.

In certain embodiments where the thiol-containing residue is an aminoacid residue, the X₁ subunit can be any one of cysteine, glutathione,mercapto-propionic acid, n-acetylated cysteine and PEGylated cysteine.In embodiments where the thiol-containing group is on a non-amino acidresidue subunit, such an organic small molecule with a thiol-containinggroup, the X₁ subunit can be a thiol-alkyl, or thioacyl moieties such as3-mercaptopropyl or 3-mercaptopropionyl residues. In one embodiment, thethiol is not homocysteine.

Accordingly, and in another embodiment, the compounds described hereinhave “clinical activity to decrease parathyroid hormone level”, whichintends that the compound, upon administration to a subject, lowersplasma parathyroid hormone as measured by the cumulative PTH area underthe curve (PTH AUC) over 4 hours post administration compared to PTH AUCof a corresponding vehicle treated control subject. The plasma PTHconcentrations are measured using, for example, a commercially availableELISA kit that detects bioactive intact PTH 1-84 (see Example 3 for aspecific kit). compound with clinical activity to decrease parathyroidhormone level reduces the PTH AUC by at least 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95% compared to the PTH AUC of a corresponding vehicletreated control subject.

The studies above, and others described below, illustrate furtherembodiments of the compounds described herein, wherein the X₁ subunit insome embodiments can be modified chemically, such as by chemicalmodification to include an acetyl group, a benzoyl group, a benzylgroup, a butyl group, a natural or unnatural amino acid such asacetylated-beta-alanine or is joined by a covalent linkage to anotherthiol moiety. Peptide therapeutics may be vulnerable to attack bypeptidases. Exopeptidases are typically non-specific enzymes whichcleave amino acid residues from the amino or carboxy termini of apeptide or protein. Endopeptidases, which cleave within an amino acidsequence, can also be non-specific; however endopeptidases frequentlyrecognize particular amino sequences (recognition sites) and cleave thepeptide at or near those sites. Accordingly, modifications to thecompound to protect it from proteolytic degradation are contemplated.

One method of protecting a peptide from proteolytic degradation involveschemically modifying, or “capping,” the amino and/or carboxy termini ofthe peptides. As used herein, the terms “chemically modified” or“capped” are used interchangeably to refer to the introduction of ablocking group to a terminus or to both termini of the compound via acovalent modification. Suitable blocking groups serve to cap the terminiof the peptides without decreasing the biological activity of thepeptides. Any residue positioned at the amino or carboxy termini, orboth, of the described compounds, including the thiol-containingsubunits can be chemically modified.

In a preferred embodiment, the amino terminus of the compound ischemically modified by acetylation, to provide an N-acetyl peptide(which may be represented as “Ac-” in a structure or formula herein). Ina preferred embodiment, the carboxy terminus of the described peptides,is chemically modified by amidation to provide a primary carboxamide atthe C-terminus (which may be represented as “—NH₂” in a peptidesequence, structure or formula herein). In a preferred embodiment, boththe amino terminus and carboxy terminus are chemically modified byacetylation and amidation, respectively. However, other capping groupsare possible. For example, the amino terminus may be capped by acylationwith groups such as an acetyl group, a benzoyl group, or with natural orunnatural amino acids such as beta-alanine capped with an acetyl group,or by alkylation with groups such as a benzyl group or a butyl group, orby sulfonylation to form sulfonamides. Similarly, the carboxy terminusmay be esterified, or converted to a secondary amide, and acylsulfonamide, or the like. In some embodiments, the amino terminus or thecarboxy terminus may comprise a site for attachment of a polyethyleneglycol (PEG) moiety, i.e., the amino or carboxy termini may bechemically modified by reaction with a suitably functionalized PEG.

Protecting peptides from endopeptidases typically involvesidentification and elimination of an endopeptidase recognition site froma peptide. Protease recognition sites are well known to those ofordinary skill in the art. Thus it is possible to identify a potentialendoprotease recognition site and then eliminating that site by alteringthe amino acid sequence within the recognition site. Residues in therecognition sequence can be moved or removed to destroy the recognitionsite. Preferably, a conservative substitution is made with one or moreof the amino acids which comprise an identified protease recognitionsite.

A. Additional Structure-Activity Relationship Studies

Additional structure activity studies were conducted, to furtherevaluate the effect of properties of each subunit in the compound on itstherapeutic activity. These studies are now to be described withreference to Example 5.

A series of compounds having an L-amino acid residue substituted for aD-amino acid residue were prepared based on the PTH-lowering scaffoldAc-c(C)arrrar-NH₂ (SEQ ID NO:3). The compounds were administered tosubjects and plasma PTH levels were assessed prior to dosing and 1, 2, 3and 4 hours after dosing, as described in Example 5 and the AUC wascalculated as the sum of the PTH concentration values at the time pointsof 1, 2, 3 and 4 hours, normalized by the AUC for the saline control atthe same time points, multiplied by 100. The results are shown in Table4.

TABLE 4 Effect of L-Amino Acid Substitution on Potency In vivo activityin normal rats 0.5 mg/kg IV bolus % PTH AUC* Compound Name Structure ofsaline control SEQ ID NO: 3 Ac-c(C)arrrar-NH₂ 8 SEQ ID NO: 34Ac-C(C)arrrar-NH₂ 17 SEQ ID NO: 35 Ac-c(C)Arrrar-NH₂ 68 SEQ ID NO: 36Ac-c(C)aRrrar-NH₂ 87 SEQ ID NO: 37 Ac-c(C)arRrar-NH₂ 182 SEQ ID NO: 38Ac-c(C)arrRar-NH₂ 130 SEQ ID NO: 39 Ac-c(C)arrrAr-NH₂ 129 SEQ ID NO: 40Ac-c(C)arrraR-NH₂ 142 Saline 100 *PTH reduction following 0.5 mg/kg IVadministration in isoflurane-anesthetized normal rats - PTH was measuredat 1, 2, 3 and 4 hours post administration and cumulative AUC wascalculated. PTH data were calculated according to the following formula:AUC_(cmpd treated)/AUC_(saline control) * 100.

The exemplary compounds shown in Table 4 were chemically modified atboth the N-terminus and the C-terminus, as indicated by the Ac and NH₂designations. The sequence of seven subunits carrrar (SEQ ID NO:3),wherein all subunits were D-amino acid residues, was modified byreplacing one subunit at a time with an L-amino acid. The X₁ subunit wasa D-Cys residue (or L-Cys residue in SEQ ID NO:34) conjugated via adisulfide linkage to an L-Cys residue, as indicated by the parentheticaldesignation (C). The PTH-lowering in vivo data in Table 4 shows thatchirality of Arg and Ala affect activity of the compounds. In oneembodiment, a compound of the sequence X₁—X₂—X₃—X₄—X₅—X₆—X₇ iscontemplated, where at least the subunits identified as X₄ and X₇ areD-amino acid residue subunits. In another embodiment, the subunitsidentified as X₄, X₅, X₆ and X₇ are D-amino acid residue subunits. In apreferred embodiment, the subunits identified as X₃, X₄, X₅, X₆ and X₇are D-amino acid residue subunits. In most preferred embodiments, thesubunits identified as X₂, X₃, X₄, X₅, X₆ and X₇ are D-amino acidresidue subunits, and all of the subunits X₁, X₂, X₃, X₄, X₅, X₆ and X₇are D-amino acid residue subunits.

In other studies, it also was found that substitution of a peptidehaving all L-amino acids with all D-amino acids did not reduce the invitro activity of the peptides tested; in fact, peptides composedentirely of D-amino acids appeared to enhance the potency for activationof the CaSR. It was also shown that some of the cationic (arginine)residues, at specific positions relative to the cysteine residue, couldbe substituted with uncharged (alanine) residues with minimal effect onthe activity toward the CaSR.

To further characterize the relationship between structure and activityagainst the CaSR, a variety of cationic peptides with different numbers(4 to 8) of arginine residues (all of which contained an N-terminalcysteine) were tested using the HEK-293 in vitro cell assay. A directcorrelation was found between the number of cationic subunits and thepotency of the compound, where potency is evidenced by ability toactivate the CaSR. Reducing the number of cationic (e.g., arginine)subunits from 5 to 4 resulted in the largest shift in potency (>10-fold)suggesting that there may be an activity inflection point betweencompounds having these net charges, that a cationic subunit at subunitX₅ is preferred for activity. Accordingly, the compounds of thestructure X₁—X₂—X₃—X₄—X₅—X₆—X₇ are contemplated, wherein X₅ is acationic subunit. In certain embodiments the X₁ is a subunit comprises athiol group that is capable of reacting with another thiol group underphysiologic conditions (a “reactive thiol”, intending a thiol thatreacts with another thiol (e.g., cysteine with cysteine) underphysiologic conditions of pH 7.4 and body temperature).

Unexpectedly, Ac-crrrrrr-NH₂ (SEQ ID NO:6) with six cationic residues,when evaluated in vivo, exhibited greater and more prolonged activitythan Ac-crrrrrrrr-NH₂ (SEQ ID NO:41), which has eight cationic residues.This is in contrast to the observation that SEQ ID NO:41 was more potentat activating the CaSR in this in vitro cell assay. Without wishing tobe bound by theory, it is thought that the superior performance ofAc-crrrrrr-NH₂ (SEQ ID NO:6) in vivo may stem from betterpharmacokinetic properties of Ac-crrrrrr-NH₂ (SEQ ID NO:6), becauseAc-crrrrrrrr-NH₂ (SEQ ID NO:6441 is expected to be taken up into cellsby virtue of its cell-penetrating characteristic, and thus removed fromproximity to the active portion of the CaSR.

To further explore the structure-activity relationship of Ac-crrrrrr-NH₂(SEQ ID NO:6), some of the cationic (arginine) residues were replacedwith uncharged (alanine) residues. It was found that replacing thecationic (arginine) residues at subunit positions X₂ and X₄ resulted ina compound (SEQ ID NO:15) that had significantly reduced potency invitro in activating the CaSR. By contrast, replacing the cationic(arginine) residues at subunit positions X₂ and X₆ resulted in acompound (SEQ ID NO:26) that retained much of the potency seen withAc-crrrrrr-NH₂ (SEQ ID NO:6). These results suggest that the position ofcharged residues in the compound contributes to potency and, in someembodiments, may outweigh the contribution of total positive charge ofthe peptide. It also appears that cationic (arginine) residues atcertain positions, such as subunit position X₅, contributedisproportionately to potency.

It was found that the presence of an N-terminal cysteine markedlyenhances the potency of the peptides for activating the CaSR. The CaSRis a 7-transmembrane G-protein-coupled receptor with a largeextracellular domain that functions as a homodimeric receptor. There are18 cysteine residues in the extracellular domain, some of which havebeen shown by polymorphism or mutational analysis to be important forreceptor activity. Of particular note are cysteines 129 and 131 of theLoop 2 region of the extracellular domain. Cysteines 129 and 131 arethought to form an intermolecular disulfide bridge between the twomonomers of the receptor complex, which is in a closed or inhibitedconfiguration. Mutation of cysteine 129 activates the CaSR, as do anumber of other mutations including a full deletion of the Loop2 region.The enhanced potency provided by the N-terminal cysteine residue in thedescribed compounds could result from a specific interaction with one ormore of the cysteine residues in the extracellular domain of the CaSR.

To further evaluate the effect of chirality of amino acid substitutionson in vitro CaSR activity, a series of analogs of Ac-crrrrrr-NH₂ (SEQ IDNO:6) were generated containing L-amino acid or achiral amino acid(glycine) substitutions at various positions and tested for potencyagainst the CaSR. Tested analogs included Ac-cGrrrGr-NH₂ (SEQ ID NO:42),(ii) Ac-cArrrAr—NH₂ (SEQ ID NO:43), and (iii) Ac-CaRrRaR-NH₂ (SEQ IDNO:44). All of the foregoing analogs had significantly lower potencythan Ac-crrrrrr-NH₂ (SEQ ID NO:6), ranging from a 10-fold difference forSEQ ID NO:44 (the most potent of the three analogs) and a more than2000-fold difference for SEQ ID NO:43 (the least potent of the threeanalogs). Ac-carrrar-NH₂ (SEQ ID NO:26), in which cationic D-amino acidresidues (D-arginine residues) at positions 2 and 6 of SEQ ID NO:6 werereplaced by uncharged D-amino acid residues (D-arginine residues), thechange in activity was much less (−3 fold difference). Thus,surprisingly, it was found that interrupting the all D-amino acidresidue of Ac-crrrrrr-NH₂ (SEQ ID NO:6) with two or more L-amino acidresidues resulted in a significant reduction in potency. Also surprisingwas that potency was decreased more than 80-fold when the interruptingresidue was an uncharged achiral amino acid residue (glycine residue)compared to when it was an uncharged L-amino acid residue (L-alanineresidue).

Also surprising was that replacing the two uncharged D-amino acidresidues (D-alanine residues) of Ac-carrrar-NH₂ (SEQ ID NO:26) withtheir L-counterparts (SEQ ID NO:43), resulted in a greater than 600-folddecrease in potency, while replacing them with an uncharged achiralamino acid residue (glycine residue) (SEQ ID NO:42) resulted in lessthan an 8-fold reduction in potency; and that replacing three cationicD-amino acid residues (D-arginine residues) of Ac-carrrar-NH₂ (SEQ IDNO:26) with their L-counterparts (SEQ ID NO:44), resulted in less than a4-fold difference in potency.

The activity of a variety of peptides and conjugates was tested againstthe human CaSR. These studies were conducted by measuring IP₁ productionin HEK293 cells that express the human CaSR. The EC₅₀ values are shownin Table 5. Each peptide was tested in eight different concentrations,in duplicates, to establish a dose response curve. Curve fitting wasperformed using GraphPad Prism. In Table 5, and thoughout thespecification, residues provided in capital letters are L-amino acids,while lower case letters indicate D-amino acids. “Ac” indicates anacetyl capping group, “NH₂” indicates an amide capping group, “Ac-bAla”is an acetylated beta-alanine, “GSH” indicates reduced glutathione, “GS”indicates oxidized glutathione, “PEG” refers to polyethylene glycol,“PEG2” and “PEGS” refer to polyethylene glycol moieties of 2 kDa and 5kDa, respectively, and “Mpa” refers to mercaptopropionic acid. A groupbracketed by parentheses indicates that group or moiety is attached tothe side-chain of the preceding subunit or amino acid residue.

TABLE 5 EC₅₀ values for cationic peptides in   CaSR in vitro assayCompound Name Structure EC₅₀ (μM) (SEQ ID NO: 45)    CHDAPIGYD 21    |(SEQ ID NO: 47) Ac-CYGRKKRRQRRR—NH₂ (SEQ ID NO: 46)    CPDYHDAGI 21    |(SEQ ID NO: 47) Ac-CYGRKKRRQRRR—NH₂ (SEQ ID NO: 47) Ac-CYGRKKRRQRRR—NH₂4.5 (SEQ ID NO: 48) Ac-YGRKKRRQRRR—NH₂ 16 (SEQ ID NO: 41)Ac-crrrrrrrr-NH₂ 0.3 (SEQ ID NO: 6)  Ac-crrrrrr-NH₂ 0.5 (SEQ ID NO: 15)Ac-cararrr-NH₂ 13 (SEQ ID NO: 26) Ac-carrrar-NH₂ 1.6 (SEQ ID NO: 4)Ac-crrrr-NH₂ 16 (SEQ ID NO: 5) Ac-crrrrr-NH₂ 2.5 (SEQ ID NO: 7)Ac-crrrrrrr-NH₂ 0.6 (SEQ ID NO: 49) Ac-caraarrr-NH₂ 1000 (SEQ ID NO: 8)Ac-carrrrr-NH₂ 1.1 (SEQ ID NO: 9) Ac-crarrrr-NH₂ 1 (SEQ ID NO: 10)Ac-crrarrr-NH₂ 1.1 (SEQ ID NO: 50) Ac-cygrkkrrqrrr-NH₂ 2 (SEQ ID NO: 51)H₂N-crrrrrr-NH₂ 0.44     | H₂N-crrrrrr-NH₂ (SEQ ID NO: 3)Ac-c(C)arrrar-NH₂ 10 (SEQ ID NO: 52) Ac-carrrar-NH₂ 0.7    |Ac-carrrar-NH₂ (SEQ ID NO: 30) Ac-bAla-crrrrrr-NH₂ 1 (SEQ ID NO: 53)Ac-c(GS)rrrrrr-NH₂ 7.8 (SEQ ID NO: 54) GS-crrrrrr — (SEQ ID NO: 55)Ac-c(Ac-C)arrrar-NH₂ 21 (SEQ ID NO: 56) Ac-c(Mpa)arrrar-NH₂ 21(SEQ ID NO: 57) Ac-c(PEG2-C)arrrar-NH₂ 2.3 (SEQ ID NO: 58)Ac-c(PEG5-C)rrrrrr-NH₂ 0.58 (SEQ ID NO: 59) Ac-c(PEG2-C)rrrrrr-NH₂ 0.02(SEQ ID NO: 34) Ac-C(C)arrrar-NH₂ 2.5 (SEQ ID NO: 60) c(C)arrrar-NH₂ 3.1(SEQ ID NO: 61) Ac-bAla-c(C)arrrar-NH₂ 2.6 (SEQ ID NO: 62)bAla-c(C)arrrar — (SEQ ID NO: 42) Ac-cGrrrGr-NH₂ 12 (SEQ ID NO: 63)Ac-cGrrrGr — (SEQ ID NO: 64) Ac-cArrrAr — (SEQ ID NO: 43)Ac-cArrrAr-NH₂ >1000 (SEQ ID NO: 44) Ac-CaRrRaR-NH₂ 5.6 (SEQ ID NO: 65)Ac-cvrrrvr-NH₂ 35 (SEQ ID NO: 66) Ac-cvrrrvr — (SEQ ID NO: 67)Ac-Crrrrrr-NH₂ 6.2 (SEQ ID NO: 68) Ac-carrrer-NH₂ 62 (SEQ ID NO: 69)Ac-cerrrar-NH₂ 31 (SEQ ID NO: 72) Ac-cakrrar-NH₂ 35 (SEQ ID NO: 73)Ac-carkrar-NH₂ 31 (SEQ ID NO: 74) Ac-carrrar-OH 31 (SEQ ID NO: 11)Ac-crrrarr-NH₂ 5.9 (SEQ ID NO: 12) Ac-crrrrar-NH₂ 0.45 (SEQ ID NO: 13)Ac-crrrrra-NH₂ 1.1 (SEQ ID NO: 75) Ac-CARRRAR-NH₂ 58 (SEQ ID NO: 76)Ac-caarrrrrr-NH₂ 4.5 (SEQ ID NO: 77) Ac-caaarrrrrr-NH₂ 4.6(SEQ ID NO: 78) Ac-carararar-NH₂ 5.3 (SEQ ID NO: 29) Ac-arrrar-NH₂ >1000(SEQ ID NO: 79) Ac-carrrarar-NH₂ 13 (SEQ ID NO: 80) crrrrrr-NH₂ 1.1(SEQ ID NO: 32) Ac-dHcy rrrrrr-NH₂ 2 (SEQ ID NO: 81)Ac-c(Benzoyl)rrrrrr-NH₂ 3.6 (SEQ ID NO: 82) Ac-c(acetyl)rrrrrr-NH₂  4.1

In another study of the structure activity relationship, thecontribution of non-cationic amino acids to the potency of the peptideswas evaluated by preparing a series of peptides with various D-aminoacid residues or glycine (Table 6) or with sterically-hinderednon-natural amino acids (Table 7), substituted at various positions inthe peptide Ac-carrrar-NH₂ (SEQ ID NO:26) and in the peptideAc-crrarar-NH₂ (SEQ ID NO:153). The peptides were administered as an IVbolus to normal Sprague Dawley rats at a dose of 0.5 mg/kg. Anintravenous (IV) bolus of saline was used as a control. Plasma PTHlevels were assessed prior to dosing and 1, 2, 3 and 4 hours afterdosing. The results are shown in the tables below, and indicate that: 1)a small amino acid such as alanine, glycine or serine is preferred atposition 6 in the Ac-carrrar-NH₂ peptide (SEQ ID NO:26), and 2) thealanine in position 2 in Ac-carrrar-NH₂ (SEQ ID NO:26) is much morepermissive to substitutions and can be substituted with hydrophobic(e.g. D-Val, D-Leu), aromatic (e.g. D-Phe), or polar (e.g. D-Ser, D-Gln)natural amino acids as well as non-natural bulky hydrophobic amino acids(e.g. dNle, dNva) but not acidic ones, and that 3) the alanine residuein position 4 of the Ac-crrarar-NH₂ (SEQ ID NO:25) peptide is also verypermissive to substitutions and can accommodate most types of naturalamino acids (as well as non-natural bulky hydrophobic amino acids (e.g.dNle, dNva) but is not permissive to amino acids that affect secondaryconformation, namely glycine or proline or amino acids with acidic sidechain.

TABLE 6 Activity of Exemplary Peptide Compounds In vivo activity innormal rats** 0.5 mg/kg IV bolus Compound % PTH AUC of SEQ ID NO.Structure* saline control Saline Saline 100 SEQ ID NO: 83 Ac-carrrfr-NH₂177 SEQ ID NO: 84 Ac-carrrir-NH₂ 161 SEQ ID NO: 85 Ac-carrrlr-NH₂ 140SEQ ID NO: 68 Ac-carrrer-NH₂ 81 SEQ ID NO: 87 Ac-carrrvr-NH₂ 79 SEQ IDNO: 88 Ac-carrrpr-NH₂ 76 SEQ ID NO: 89 Ac-carrrhr-NH₂ 48 SEQ ID NO: 90Ac-carrrqr-NH₂ 41 SEQ ID NO: 91 Ac-carrrtr-NH₂ 18 SEQ ID NO: 92Ac-carrrsr-NH₂ 6 SEQ ID NO: 93 Ac-carrrGr-NH₂ 5 SEQ ID NO: 94Ac-cerrrar-NH₂ 103 SEQ ID NO: 95 Ac-cGrrrar-NH₂ 45 SEQ ID NO: 96Ac-cirrrar-NH₂ 33 SEQ ID NO: 97 Ac-cprrrar-NH₂ 30 SEQ ID NO: 98Ac-clrrrar-NH₂ 26 SEQ ID NO: 99 Ac-cqrrrar-NH₂ 24 SEQ ID NO: 100Ac-ctrrrar-NH₂ 23 SEQ ID NO: 101 Ac-cvrrrar-NH₂ 19 SEQ ID NO: 102Ac-csrrrar-NH₂ 13 SEQ ID NO: 103 Ac-chrrrar-NH₂ 1 SEQ ID NO: 104Ac-cfrrrar-NH₂ 0 SEQ ID NO: 105 Ac-crrGrar-NH₂ 69 SEQ ID NO: 106Ac-crrprar-NH₂ 68 SEQ ID NO: 107 Ac-crrerar-NH₂ 56 SEQ ID NO: 108Ac-crrtrar-NH₂ 13 SEQ ID NO: 109 Ac-crrhrar-NH₂ 9 SEQ ID NO: 110Ac-crrfrar-NH₂ 6 SEQ ID NO: 111 Ac-crrsrar-NH₂ 4 SEQ ID NO: 112Ac-crrqrar-NH₂ 4 SEQ ID NO: 113 Ac-crrvrar-NH₂ 3 SEQ ID NO: 114Ac-crrlrar-NH₂ 1 SEQ ID NO: 115 Ac-crrirar-NH₂ 0 *Bolded font indicatesrespective substitution of alanine residues in Ac-carrrar-NH₂ (SEQ IDNO: 6) or Ac-crrarar-NH₂ (SEQ ID NO: 25). **PTH reduction following 0.5mg/kg IV administration in isoflurane-anesthetized normal rats - PTH wasmeasured at 1, 2, 3 and 4 hours post administration and cumulative AUCwas calculated. PTH data were calculated according to the followingformula: AUC_(cmpd treated)/AUC_(saline control) * 100.

TABLE 7 Activity of Exemplary Peptide Compounds In vivo activity innormal rats* 0.5 mg/kg IV bolus % PTH AUC of SEQ ID NO. CompoundStructure* saline control** Saline Saline 100 SEQ ID NO: 116Ac-crr-Sar-rar-NH₂ 141 SEQ ID NO: 117 Ac-carrr-Sar-r-NH₂ 111 SEQ ID NO:118 Ac-c-Nma-rrr-Nma-r-NH₂ 105 SEQ ID NO: 119 Ac-crrar-Nma-r-NH₂ 101 SEQID NO: 120 Ac-c-Aib-rrr-Aib-r-NH₂ 94 SEQ ID NO: 121 Ac-crr-Nma-rar-NH₂86 SEQ ID NO: 122 Ac-carrr-Nma-r-NH₂ 74 SEQ ID NO: 123Ac-c-Aib-rrrar-NH₂ 70 SEQ ID NO: 124 Ac-carrr-Aib-r-NH₂ 68 SEQ ID NO:125 Ac-c-Sar-rrr-Sar-r-NH₂ 65 SEQ ID NO: 126 Ac-crrar-Sar-r-NH₂ 62 SEQID NO: 127 Ac-c-Nma-rrrar-NH₂ 56 SEQ ID NO: 128 Ac-c-Sar-rrrar-NH₂ 50SEQ ID NO: 129 Ac-carrr-Nle-r-NH₂ 64 SEQ ID NO: 130Ac-c-dNle-rrr-dNle-r-NH₂ 54 SEQ ID NO: 131 Ac-carrr-dNva-r-NH₂ 54 SEQ IDNO: 132 Ac-c-dNva-rrr-dNva-r-NH₂ 27 SEQ ID NO: 133 Ac-crrar-dNle-r-NH₂26 SEQ ID NO: 134 Ac-c-dNle-rrrar-NH₂ 10 SEQ ID NO: 135Ac-crrar-dNva-r-NH₂ 8 SEQ ID NO: 136 Ac-c-dNva-rrrar-NH₂ 7 SEQ ID NO:137 Ac-crr-dNva-rar-NH₂ 3 SEQ ID NO: 138 Ac-crr-dNle-rar-NH₂ 3 *Boldedfont indicates respective substitution of alanine residues inAc-carrrar-NH₂ (SEQ ID NO: 26) or Ac-crrarar-NH₂ (SEQ ID NO: 25). Sar =the non-natural amino acid Sarcosine; Nma = N-methyl alanine; AiB =amino isobutyric acid; dNva = D-Norvaline; dNle = D-Norleucine **PTHreduction following 0.5 mg/kg IV administration inisoflurane-anesthetized normal rats - PTH was measured at 1, 2, 3 and 4hours post administration and cumulative AUC was calculated. PTH datawere calculated according to the following formula:AUC_(cmpd treated)/AUC_(saline control) * 100.

TABLE 8 Activity of Exemplary Peptide Compounds In vivo activity innormal rats** 0.5 mg/kg IV bolus % PTH AUC of saline SEQ ID NO.Compound Structure* control Saline Saline 100 SEQ ID NO: 97Ac-c(C)arrrar-NH₂ 8 SEQ ID NO: 101 Ac-c(GS)rrrrrr-NH₂ 12 SEQ ID NO: 139Ac-c(dHcy)arrrar-NH₂ 32 SEQ ID NO: 140 Ac-c(Mpa)arrrar-NH₂ 25SEQ ID NO: 141 Ac-c(Ac-C)arrrar-NH₂ 38 SEQ ID NO: 142 Ac-c(c)arrrar-NH₂0 SEQ ID NO: 143*** Ac-c(C-PEG20)rrrrrr-NH₂ 25 SEQ ID NO: 144****Ac-c(C-PEG40)rrrrrr-NH₂ 15 SEQ ID NO: 145    CEEEEEE 40    |Ac-crrrrrr-NH2 SEQ ID NO: 145  CEEEEEE 42  | SEQ ID NO: 26Ac-carrrar-NH2 SEQ ID NO: 25 Ac-crrarar-NH2 2    | SEQ ID NO: 25Ac-crrarar-NH2 SEQ ID NO: 26 Ac-carrrar-NH2 1    | SEQ ID NO: 26Ac-carrrar-NH2 *Bolded font showing in parenthesis indicates respectivethiol-containing conjugating groups.GS = oxidized glutathione; dHcy =D-homocysteine; Mpa = Mercaptopropionic acid; PEG = polyethylene glycol.**PTH reduction following 0.5 mg/kg IV administration inisoflurane-anesthetized normal rats - PTH was measured at 1, 2, 3 and 4hours post administration and cumulative AUC was calculated. PTH datawere calculated according to the following formula:AUC_(cmpd treated)/AUC_(saline control)*100. ***Compound was dosed at 10mg/kg (~ equivalent molarity to a 0.5 mg/kg non-PEGylated peptide)****Compound was dosed at 20 mg/kg (~ equivalent molarity to a 0.5 mg/kgnon-PEGylated peptide)B. Histamine Response and Structure-Activity Relationship Studies

Poly-cationic compounds have been reported in the literature to triggerthe release of the active biogenic amine histamine. See Church et al.,J. Immunol., 128(5):2116-2121 (1982); Lagunoff et al., Ann. Rev.Pharmacol. Toxicol., 23:331-51 (1983). It is thought that histaminerelease is a result of mast cell and basophil activation occurring in aGai dependent manner. See Aridor et al., J. Cell Biol., 111(3):909-17(1990). Reducing or eliminating this physiological reaction isdesirable, inter alia, for improving the therapeutic margin of cationicpeptide calcimimetics for the treatment of SHPT.

Studies were conducted to evaluate the histamine release induced upon invivo administration of the compounds described herein. In a first study,described in Example 6, dosing by IV bolus or infusion into normalSprague Dawley rats was used to evaluate histamine release associatedwith various compounds. To evaluate the effect of net positive charge onthe histamine release associated with a compound, peptides containing 4to 7 cationic (arginine) residues were generated and tested for theirability to trigger histamine release in vivo, according to the proceduredescribed in Example 6. The tested peptides included (i) Ac-crrrr-NH₂(SEQ ID NO:4), (ii) Ac-crrrrr-NH₂ (SEQ ID NO:5), (iii) Ac-crrrrrr-NH₂(SEQ ID NO:6) and (iv) Ac-crrrrrrrr-NH₂ (SEQ ID NO:41).

As shown in FIG. 3, when an equivalent number of moles of each peptidewas administered by IV bolus to normal rats, SEQ ID NO:41 (8 arginineresidues) displayed the largest induction of histamine. Other compoundswith fewer Arg residues, including SEQ ID NO:6 (6 arginine residues),SEQ ID NO:5 (5 arginine residues), and SEQ ID NO:4 (4 arginineresidues), also produced a spike in histamine level, but to a lesserextent compared to SEQ ID NO:41. SEQ ID NO:6, SEQ ID NO:5 and SEQ IDNO:4 generated milder responses in their histamine release activity(˜2-3 fold above baseline). SEQ ID NO:5 and SEQ ID NO:4 were, however,less potent than SEQ ID NO:6 with respect to lowering plasma PTH.

Because the PTH-reducing activity of Ac-crrrrrr-NH₂ (SEQ ID NO:6) wasaccompanied by lack of a histamine response, additional evaluations wereconducted based on Ac-crrrrrr-NH₂ (SEQ ID NO:6) in order to evaluatewhether it was possible to still further decrease histamine responsewithout sacrificing PTH-lowering activity. As will be shown in the databelow, substitution of cationic (arginine) residues in Ac-crrrrrr-NH₂(SEQ ID NO:6) with non-cationic (alanine) residues was performed toproduce a series of analogs with an overall reduced net charge andreduced charge density. Of these analogs, both Ac-cararrr-NH₂ (SEQ IDNO:15) and Ac-carrrar-NH₂ (SEQ ID NO:26) were associated with lack of ahistamine response when administered to rats by IV bolus. Importantly,these two peptides retained their potent calcimimetic properties andwere able to reduced PTH secretion in both normal rats and rats withrenal dysfunction.

The compound Ac-crrrrrr-NH₂ identified as SEQ ID NO:6 (2.1 μmole/kg=2.3mg/kg) triggered an observable histamine response of about 2-3 fold overbaseline compared to 6-9 fold with SEQ ID NO:41 when dosed by IV bolus(given over less than 1 minute) in normal rats. The histamine releasetriggered by Ac-crrrrrr-NH₂ (SEQ ID NO:6) peaked at 5 minutes afterdosing and returned to baseline levels 15 minutes later (FIG. 3).Further reduction in the number of charged subunits to 5 and 4 arginineresidues per peptide (SEQ ID NO:5 and SEQ ID NO:4, respectively) furtherreduced the histamine response as compared with the longeroligo-arginine peptides; however, a 2-3 fold increase in histamine overbaseline was still observed 5 minutes after IV bolus dosing (FIG. 3).These results suggest a relationship between the net charge of thepeptide and the associated release of histamine. It is also noted thatarginine-rich peptides with fewer than 7 arginines are quite limited intheir ability to enter cells, suggesting that cell penetration is notrequired to trigger histamine release.

The histamine release associated with the PTH-lowering compoundsAc-crrrrrr-NH₂ (SEQ ID NO:6) and Ac-c(C)arrrar-NH₂ (SEQ ID NO:3) wasevaluated in vivo. The compound Ac-c(C)arrrar-NH₂ (SEQ ID NO:3) has thefollowing structure:

(SEQ ID NO: 3)    C       | Ac-carrrar-NH₂This conjugate structure is denoted herein as Ac-c(C)arrrar-NH₂ (SEQ IDNO:273 where the L-Cys residue linked to the thiol-containing residue inthe X₁ subunit of the compound (here, a D-Cys residue) via a Cys-Cysdisulfide bond, is placed in parenthesis in the formula. This notationis used throughout to designate that the parenthetical moiety is linkedto a second thiol-containing group. Relative to Ac-crrrrrr-NH₂ (SEQ IDNO:6) the compound Ac-c(C)arrrar-NH₂ (SEQ ID NO: 3) has two cationic(arginine) residues substituted with uncharged (alanine) residues atsubunit positions X₂ and X₆. In addition, the D-Cys residue in the X₁position is conjugated to an L-Cys residue.

These two compounds were administered to isoflurane-anesthetized rats(Sprague Dawley) at 3 mg/kg by intravenous (IV) bolus (given over lessthan 1 minute). Blood was drawn prior to dosing and a 5, 15 and 30minutes after dosing. Histamine concentration was measured, and the foldchange in blood histamine concentration relative to the pre-dose bloodhistamine concentration is shown in FIG. 4. The compound Ac-crrrrrr-NH₂(SEQ ID NO:6, open bars) induced a histamine response, observed at thedata point 5 minutes post-dosing where a 7-fold increase in histaminelevel was observed. The compound Ac-c(C)arrrar-NH₂ (SEQ ID NO:3, crosshatched bars) induced no apparent histamine response, as seen by thedata points at 5, 10 and 15 minutes post dosing where the histaminelevel was not increased relative to the pre-dose (time zero) histaminelevel.

To further evaluate the relationship between compound structure andhistamine release, a series of compounds was prepared and assessed fortheir ability to trigger induction of histamine in an in vitro assayusing rat peritoneal mast cells. In this assay, compounds are incubatedat 10 μM for 15 minutes at 37° C. with cells isolated from peritoneallavage of SD rats. Following incubation, cell medium is collected andhistamine is determined. The data is shown in Table 9.

TABLE 9 In vitro Histamine Induction in Rat PeritonealMast Cells of Exemplary Peptide Compounds *Histamine in vitro Foldchange of non- SEQ ID NO. Compound Sequence treated at 10uM* SalineSaline 1.0 SEQ ID NO: 6 Ac-crrrrrr-NH₂ 11.5 SEQ ID NO: 8 Ac-carrrrr-NH₂6.6 SEQ ID NO: 9 Ac-crarrrr-NH₂ 6.8 SEQ ID NO: 10 Ac-crrarrr-NH₂ 5.3SEQ ID NO: 11 Ac-crrrarr-NH₂ 5.0 SEQ ID NO: 12 Ac-crrrrar-NH₂ 5.0SEQ ID NO: 13 Ac-crrrrra-NH₂ 4.1 SEQ ID NO: 15 Ac-cararrr-NH₂ 2.5SEQ ID NO: 22 Ac-caarrrr-NH₂ 1.2 SEQ ID NO: 17 Ac-crraarr-NH₂ 1.3SEQ ID NO: 146 Ac-crrrraa-NH₂ 1.9 SEQ ID NO: 26 Ac-carrrar-NH₂ 1.4SEQ ID NO: 3 Ac-c(C)arrrar-NH₂ 0.6 SEQ ID NO: 16 Ac-carrarr-NH₂ 1.4SEQ ID NO: 19 Ac-carrrra-NH₂ 1.3 SEQ ID NO: 23 Ac-crarrar-NH₂ 1.5SEQ ID NO: 18 Ac-crararr-NH₂ 1.4 SEQ ID NO: 20 Ac-crarrra-NH₂ 1.1SEQ ID NO: 25 Ac-crrarar-NH₂ 1.2 SEQ ID NO: 14 Ac-crrarra-NH₂ 1.6SEQ ID NO: 130 Ac-c-dNle-rrr-dNle-r-NH₂ 9.2 SEQ ID NO: 132Ac-c-dNya-rrr-dNya-r-NH₂ 4.1 SEQ ID NO: 28 Ac-c(C)rrarar-NH₂ 0.7SEQ ID NO: 24 Ac-craarrr-NH₂ 1.0 SEQ ID NO: 21 Ac-crrraar-NH₂ 1.0SEQ ID NO: 134 Ac-c-dNle-rrrar-NH₂ 2.2 SEQ ID NO: 129Ac-carrr-dNle-r-NH₂ 2.6 SEQ ID NO: 136 Ac-c-dNva-rrrar-NH₂ 2.1SEQ ID NO: 131 Ac-carrr-dNva-r-NH₂ 1.8 SEQ ID NO: 133Ac-crrar-dNle-r-NH₂ 4.3 SEQ ID NO: 135 Ac-crrar-dNva-r-NH₂ 1.1SEQ ID NO: 95 Ac-cGrrrar-NH₂ 1.5 SEQ ID NO: 99 Ac-cqrrrar-NH₂ 1.9SEQ ID NO: 103 Ac-chrrrar-NH₂ 1.6 SEQ ID NO: 96 Ac-cirrrar-NH₂ 3.0SEQ ID NO: 98 Ac-clrrrar-NH₂ 2.2 SEQ ID NO: 97 Ac-cprrrar-NH₂ 0.8SEQ ID NO: 102 Ac-csrrrar-NH₂ 0.9 SEQ ID NO: 100 Ac-ctrrrar-NH₂ 1.1SEQ ID NO: 101 Ac-cvrrrar-NH₂ 1.5 SEQ ID NO: 93 Ac-carrrGr-NH₂ 0.9SEQ ID NO: 90 Ac-carrrqr-NH₂ 0.9 SEQ ID NO: 89 Ac-carrrhr-NH₂ 2.0SEQ ID NO: 84 Ac-carrrir-NH₂ 1.8 SEQ ID NO: 85 Ac-carrrlr-NH₂ 2.5SEQ ID NO: 88 Ac-carrrpr-NH₂ 1.0 SEQ ID NO: 92 Ac-carrrsr-NH₂ 1.2SEQ ID NO: 91 Ac-carrrtr-NH₂ 1.7 SEQ ID NO: 87 Ac-carrrvr-NH₂ 1.2SEQ ID NO: 147 Ac-cakkkak-NH₂ 1.1 SEQ ID NO: 72 Ac-cakrrar-NH₂ 1.1SEQ ID NO: 73 Ac-carkrar-NH₂ 1.4 SEQ ID NO: 105 Ac-crrGrar-NH₂ 1.8SEQ ID NO: 112 Ac-crrqrar-NH₂ 1.2 SEQ ID NO: 109 Ac-crrhrar-NH₂ 2.3SEQ ID NO: 115 Ac-crrirar-NH₂ 3.4 SEQ ID NO: 114 Ac-crrlrar-NH₂ 4.5SEQ ID NO: 106 Ac-crrprar-NH₂ 1.1 SEQ ID NO: 111 Ac-crrsrar-NH₂ 2.0SEQ ID NO: 108 Ac-crrtrar-NH₂ 1.2 SEQ ID NO: 113 Ac-crrvrar-NH₂ 1.9SEQ ID NO: 104 Ac-cfrrrar-NH₂ 6.8 SEQ ID NO: 83 Ac-carrrfr-NH₂ 4.0SEQ ID NO: 68 Ac-carrrer-NH₂ 1.3 SEQ ID NO: 110 Ac-crrfrar-NH₂ 6.2SEQ ID NO: 107 Ac-crrerar-NH₂ 0.6 SEQ ID NO: 86 Ac-carrkar-NH₂ 1.0SEQ ID NO: 70 Ac-carrrak-NH₂ 0.9 SEQ ID NO: 148 Ac-cararar-NH₂ 0.6SEQ ID NO: 25 Ac-crrarar-NH₂ 9.3    | Ac-carrrar-NH₂ SEQ ID NO: 149Ac-crrarGr-NH₂ 1.6 SEQ ID NO: 150 Ac-crrarqr-NH₂ 1.6 SEQ ID NO: 151Ac-crrarhr-NH₂ 2.5 SEQ ID NO: 152 Ac-crrarir-NH₂ 4.1 SEQ ID NO: 153Ac-ca(DAP)rrar-NH₂ 1.6 SEQ ID NO: 154 Ac-ca(dHar)(dHar)(dHar)ar-NH₂ 2.7*Method set forth in Example 7 Abbreviations: See Example 7

To further evaluate the relationship between compound structure andhistamine release, a series of compounds was prepared assessed for theirability to trigger induction of histamine in an in vivo assays. The datais shown in Table 10.

TABLE 10 In vivo Histamine Induction of Exemplary Peptide Compounds *Histamine response in vivo Fold change from pre-dose levels 5′ postinjection SEQ ID NO. Compound Structure 2 mg/kg IV bolus Saline Saline1.0 SEQ ID NO: 6 Ac-crrrrrr-NH₂ 2.7 SEQ ID NO: 26 Ac-carrrar-NH₂ 1.0 SEQID NO: 25 Ac-crrarar-NH₂ 0.9 SEQ ID NO: 15 Ac-cararrr-NH₂ 1.0 SEQ ID NO:18 Ac-crararr-NH₂ 1.1 SEQ ID NO: 20 Ac-crarrra-NH₂ 1.0 SEQ ID NO: 19Ac-carrrra-NH₂ 0.9 SEQ ID NO: 23 Ac-crarrar-NH₂ 0.8 SEQ ID NO: 18Ac-crararr-NH₂ 1.0 SEQ ID NO: 27 Ac-c(C)arrrar-NH₂ 0.9 SEQ ID NO: 28Ac-c(C)rrarar-NH₂ 0.9 * Method set forth in Example 7.

Accordingly, and as can be appreciated in view of the PTH data and thehistamine data described hereinabove, in one embodiment, a compound thathas activity to decrease PTH level in a subject in the absence of ahistamine response is contemplated. In certain embodiments, absence of ahistamine response intends a dose of the compound that produces a lessthan 10-fold, more preferably 8-fold, still more preferably 5-fold, andeven still more preferably 3-fold, increase in histamine, measured invitro in an assay as described herein, where the fold change isdetermined based on histamine levels before incubation with the compoundand after 15 minutes incubation with compound. In a specific embodiment,the histamine response is determined in an in vitro assay using ratperitoneal mast cells isolated from peritoneal lavage of normal SpragueDawley rats, and where the fold change is determined based on histaminelevels before incubation with the compound and after 15 minutesincubation with compound. In the studies conducted herein, the in vitroevaluation of histamine release was performed using isolated ratperitoneal mast cells isolated by peritoneal lavage using cold HBSS+25mM HEPES pH 7.4 containing heparin (5 u/mL). Cells were washed twice instimulation buffer (HBSS+25 mM HEPES pH 7.4) and incubated with 10 μM ofcompound in stimulation buffer (HBSS+25 mM HEPES pH 7.4) for 15 minutesin a 96-well plate (106/well) at 37° C. Cell supernatant was analyzedfor histamine using histamine EIA kit (Cayman #589651).

In another embodiment, a compound that has activity to decrease PTHlevel in a subject in the absence of an clinical histamine response iscontemplated. As used herein, absence of a “clinical histamine response”intends is that a therapeutically effective amount of a compound asdescribed herein is administered to the subject without producing aclinically adverse increase in plasma or blood histamine as measured5-10 minutes after completion of dosing or over the course of treatment.For example, when a compound required to produce a desired therapeuticeffect is administered to a subject by bolus (as used herein “bolus”means administered over one minute or less) produced an increase inplasma or blood histamine 5-10 minutes after completion of dosing thatis less than 15-fold, 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold,4-fold, 3-fold, 2-fold above pre-dose levels.

As can be appreciated from the studies described above, in oneembodiment, the compound comprises a sequence of 3 to 35 amino acidresidues, wherein a plurality of positively charged amino acid residuesubunits is present in the sequence. In some embodiments, the describedcompounds comprise 5 to 25 subunits, and in a preferred embodiment eachsubunit is an amino acid residue. In other embodiments, the describedcompounds comprise 6 to 12 subunits. In still other embodiments, thedescribed compounds comprise 3 to 9 amino acid subunits. In alternativeembodiments the described compounds comprise 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, or 35 subunits.

The subunits of the described compounds are, in one embodiment,independently selected from natural or unnatural amino acids, or theiranalogs, and may have either the L- or D-configuration (except forglycine which is achiral). Glycine, the aliphatic residues alanine,valine, leucine, or isoleucine, proline, the hydroxyl residues serineand threonine, the acidic residues aspartic acid and glutamic acid, theamide residues asparagine, and glutamine, the basic residues lysine andarginine, histidine, the aromatic residues phenylalanine, tyrosine, andtryptophan, and the sulfur-containing residues methionine and cysteineare all contemplated for use in the described compounds. The number ofpositively charged subunits, and their density can affect the potency ofthe compound for reducing PTH. In some embodiments, positively chargedsubunits are separated by one or more other subunits (“separatingsubunits”). In one embodiment, the separating subunits are alanineresidues. In some embodiments, the chirality of the separating subunitaffects the potency of the compound.

Positively charged amino acid residues of the described compounds may bea specific natural or unnatural residue, or analog thereof, havingeither the L- or D-configuration (e.g., L-arginine) that is repeated inthe sequence, or may be a variety of natural or unnatural residues, oranalogs thereof, having either the L- or D-configuration. In someembodiments, the compound is a peptide comprised of from 3 to 20positively charged amino acid residues, 6 to 12 positively charged aminoacid residues, 3 to 9 positively charged amino acid residues. In someembodiments, the peptides comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 positively charged amino acid residues.

In some embodiments, the positively charged amino acid residues areindependently selected from natural amino acids. In some embodiments,the positively charged amino acid residues are independently selectedfrom natural and/or unnatural amino acids. In some embodiments, thepositively charged amino acid residues are independently selected fromthe group consisting of arginine, lysine, histidine,2,3-diaminopropionic acid (Dap), 2,4-diaminobutyric acid (Dab),ornithine, and homoarginine. In a preferred embodiment, the positivelycharged amino acid residues are arginine residues.

In some embodiments, the compound is a peptide and is a singlecontinuous peptide chain or strand. In other embodiments, the compoundis a peptide that is branched. In still other embodiments, the peptideis conjugated to one or more thiol-containing moieties (each, a“thiol-containing conjugating group” or a “conjugating group”). In apreferred embodiment, and as merely illustrative, the peptide compoundis conjugated to a Cys conjugating group, via a (—S—S—)disulfide bond(for example -Cys-Cys-). As used herein, the term “compound” is intendedto encompass both such peptides and such conjugates.

The compounds typically comprise one or more thiol moieties, preferablyone or more reactive thiol moieties. Subunits that have a thiol groupinclude non-amino acid compounds having a thiol group and amino acidswith a thiol group. The thiol group of the thiol-containing subunit maybe in a conjugated form (e.g., via a disulfide bond to a conjugatinggroup) or in an unconjugated form (i.e., as a reduced thiol). In apreferred embodiment, when the thiol group is in either an unconjugatedform or a conjugated form, it is capable of forming a disulfide bondwith a thiol-containing group. The thiol-containing residue may belocated at any position along the peptide chain, including the aminoterminus, the carboxy terminus, or some other position. In a preferredembodiment, the thiol-containing residue or subunit may be located atthe amino terminus. In other embodiments, the thiol-containing residueor subunit may be located at the carboxy terminus or within the peptidesequence.

Some representative examples of thiol-containing residues include,without limitation, cysteine, mercaptopropionic acid, homo-cysteine, andpenicillamine. When the thiol-containing residue contains a chiralcenter, it may be present in the L- or D-configuration. In a preferredembodiment, the thiol-containing residue is cysteine.

In some embodiments, the cross-linkage between the thiol containingsubunit at the X₁ position in the compound and the thiol-containingconjugating group may be cleavable and/or exchangeable with otherthiol-containing conjugating groups such as cysteine (e.g., by reductionof the disulfide linkage) in vivo to yield a biologically active form ofthe compound. In this way, the conjugate may function as a pro-drug ofthe compound. A conjugating group also may be used to modify thephysicochemical, pharmacokinetic and/or pharmacodynamic properties ofthe described compounds (e.g., conjugation via a disulfide linkage to alarge PEGylated moiety to enhance the pharmacokinetics).

In some embodiments, the compound is a peptide comprised of the aminoacid sequence(X_(aa1))—(X_(aa2))—(X_(aa3))—(X_(aa4))—(X_(aa5))—(X_(aa6))—(X_(aa7))(SEQ ID NO:155), wherein (X_(aa1)) is a thiol-containing amino acidresidue, (X_(aa2)) is a non-cationic amino acid residue, (X_(aa3)) isany amino acid residue, (X_(aa4)) is any amino acid residue, (X_(aa5))is a cationic amino acid residue, (X_(aa6)) is a non-cationic residue,and (X_(aa7)) is any amino acid residue. The peptide may be modified atthe N-terminus, the C-terminus, or both. In a preferred embodiment, thepeptide is modified at both the N-terminus and C-terminus by acetylationand amidation, respectively.

In some embodiments, a peptide comprises the amino acid sequence(D-Cys)-(X_(aa2))—(X_(aa3))—(X_(aa4))—(X_(aa5))—(X_(aa6))—(X_(aa7)) (SEQID NO:156), wherein (X_(aa2)) is a non-cationic amino acid residue,(X_(aa3)) is any amino acid residue, (X_(aa4)) is any amino acidresidue, (X_(aa5)) is selected from the group consisting of D-Arg,L-Arg, D-Lys and L-Lys, (X_(aa6)) is a non-cationic residue, and(X_(aa7)) is any amino acid residue. The peptide may have an N-terminalcap, a C-terminal cap, or both. In a preferred embodiment, the peptidehas both an N-terminal cap and a C-terminal cap.

In some embodiments, a peptide comprises the amino acid sequence(D-Cys)-(X_(aa2))—(X_(aa3))—(X_(aa4))—(X_(aa5))—(X_(aa6))—(X_(aa7)) (SEQID NO:157), wherein (X_(aa2)), (X_(aa3)) and (X_(aa4)) are,independently, any amino acid residue (but in a preferred embodimentare, independently, selected from the group consisting of D-Ala, D-Val,D-Leu, D-NorVal, and D-NorLeu), (X_(aa5)) and (X_(aa7)) are,independently, any cationic amino acid residue (but in a preferredembodiment are, independently, selected from the group consisting ofD-Arg, L-Arg, D-Lys and L-Lys), (X_(aa6)) is a non-cationic amino acidresidue (in a preferred embodiment, selected from the group consistingof D-Ala, D-Val, D-Leu, D-NorVal and D-NorLeu). The peptide may have anN-terminal cap, a C-terminal cap, or both. In a preferred embodiment,the peptide has both an N-terminal cap and a C-terminal cap.

In some embodiments, a peptide comprises the amino acid sequence(D-Cys)-(X_(aa2))—(X_(aa3))—(X_(aa4))—(X_(aa5))—(X_(aa6))—(X_(aa7)) (SEQID NO:158), wherein (X_(aa2)) is a non-cationic amino acid residue,(X_(aa3)) is any amino acid residue, (X_(aa4)) is any amino acidresidue, (X_(aa5)) is selected from the group consisting of D-Arg,L-Arg, D-Lys and L-Lys, (X_(aa6)) is a non-cationic residue, and(X_(aa7)) is any amino acid residue. The peptide may have an N-terminalcap, a C-terminal cap, or both. In a preferred embodiment, the peptidehas both an N-terminal cap and a C-terminal cap.

In some embodiments, a peptide comprises the amino acid sequence(D-Cys)-(D-Ala)-(X_(aa3))—(X_(aa4))-(D-Arg)-(D-Ala)-(X_(aa7)) (SEQ IDNO:159), wherein (X_(aa3)) is any cationic amino acid residue, (X_(aa4))is any cationic amino acid residue, and (X_(aa7)) is any cationic aminoacid residue. The peptide may have an N-terminal cap, a C-terminal cap,or both. In a preferred embodiment, the peptide has both an N-terminalcap and a C-terminal cap.

In some embodiments, a peptide comprises the amino acid sequence(D-Cys)-(X_(aa2))—(X_(aa3))-(D-Ala)-(D-Arg)-(D-Ala)-(X_(aa7)) (SEQ IDNO:160), wherein (X_(aa2)), (X_(aa3)) and (X_(aa7)) are, independently,any cationic amino acid residue. The peptide may have an N-terminal cap,a C-terminal cap, or both. In a preferred embodiment, the peptide hasboth an N-terminal cap and a C-terminal cap.

Another embodiment is a calcimimetic peptide, comprising a sequence ofamino acids linked by peptide bonds, wherein the sequence comprises 5 to10 amino acid residues, and wherein the sequence comprises an aminoterminus, a carboxy terminus, at least one thiol-containing residue, andfrom 3 to 9 positively charged residues. In one embodiment, the at leastone thiol-containing residue is a cysteine residue. In another aspect,the cysteine residue is positioned at the amino terminus of the peptide.In certain embodiment, the cysteine residue is an L-Cys residue, a D-Cysresidue, or an L- or D-homoCys residue. In other embodiments, the aminoacid residues of the peptide are D-amino acids or L-amino acids.

Also encompassed within the scope of the claimed compounds arepeptidomimetic molecules that comprise approximately seven subunits,wherein at least one subunit contains a thiol moiety, preferably areactive thiol moiety, and other subunits are a plurality ofnon-cationic subunits, and from 1 to 4 positively charged subunits. Suchpeptidomimetic molecules may comprise non-peptide bonds between two ormore of the subunits. The various features of the compounds discussedabove apply generally to the peptidomimetic molecule. For example, asdiscussed above, the subunits used to construct the molecules can benaturally-occurring amino acids, or residues with non-natural sidechains, the termini of the modules can be capped or non-capped in themanner discussed above. Similarly, the amino acid residues of themolecule can be L- or D-amino acid residues. Also as discussed above,the thiol-containing residues can be in a reduced or oxidized form withany of the thiol-containing moieties discussed above.

Many peptidomimetic frameworks and methods for their synthesis have beendeveloped (Babine, R. E.; Bender, S. L., Chem. Rev., 97:1359, 1997;Hanessian, S.; et al., Tetrahedron, 53:12789, 1997; Fletcher, M. D.;Cambell, M. C., Chem. Rev., 98:763, 1998); Peptidomimetics Protocols;Kazmierski W. M., Ed.; Methods in Molecular Medicine Series, Vol. 23;Humana Press, Inc.; Totowa, N.J. (1999).

Conjugates

In some embodiments, the compound is chemically cross-linked to athiol-containing conjugating group via a disulfide bond between thethiol of the compound and a thiol from the conjugating group. Thethiol-containing conjugating group can be a small molecule, such ascysteine, or a macromolecule, such as a polypeptide containing acysteine residue. Examples of suitable thiol-containing conjugatinggroups include cysteine, glutathione, thioalkyl, moieties such asthiobenzyl, mercaptopropionic acid, N-acetylated cysteine, cysteamide,N-acetylcysteamide, homocysteine, penicillamine and poly(ethyleneglycol) (PEG) modified (referred to as “PEGylated”) thiols such asPEGylated cysteine or a duplication of the compound (ie., to form ahomodimer linked by a disulfide linkage). In a preferred embodiment, thethiol-containing conjugating group is cysteine. Other cysteine homologsare also contemplated for use as thiol-containing conjugating groups,either alone or comprised in a larger conjugating group. Similarly,stereoisomers of cysteine, homocysteine, and cysteamide are suitable foruse as thiol-containing moieties. Conjugating groups can be used toimprove chemical stability and therefore shelf-life of a pharmaceuticalproduct. In certain embodiments the thiol-containing conjugating groupand the peptide are the same (i.e., the conjugate is a dimer), whichunexpectedly showed very good chemical stability compared toheterologous conjugating group such as cysteine. Without being bound bytheory, presumably when the thiol-containing conjugating group and thepeptide are the same, then any disproportionation (e.g., scrambling ofthe conjugating group) will reconstitute the original dimer compound. Incontrast, disproportionation of a compound with a heterologousconjugating group such as cysteine can lead to formation of homo-dimersof the peptide plus cystine (cysteine-cysteine homodimer) plus residualparent compound. A homo-dimer of the peptide (i.e., conjugating groupand the peptide are the same) would be converted to a cysteineconjugated form of the peptide in vivo due to the high concentration ofreduced cysteine in systemic circulation.

In some embodiments, the teachings include a disulfide conjugate of athiol-containing conjugating group and a peptide comprising the aminoacid sequence(X_(aa1))—(X_(aa2))—(X_(aa3))—(X_(aa4))—(X_(aa5))—(X_(aa6))—(X_(aa7))(SEQ ID NO:155), wherein (X_(aa1)) is an amino acid residue with athiol-containing moiety, (X_(aa2)) is a non-cationic amino acid residue,(X_(aa3)) is any amino acid residue, (X_(aa4)) is any amino acidresidue, (X_(aa5)) is a cationic amino acid residue, (X_(aa6)) is anon-cationic residue, and (X_(aa7)) is any amino acid residue. Thepeptide may have an N-terminal cap, a C-terminal cap, or both. In apreferred embodiment, the peptide has both an N-terminal cap and aC-terminal cap. In a preferred embodiment, the thiol-containingconjugating group is selected from the group consisting of D-Cys, L-Cys,a peptide containing D-Cys, and a peptide containing L-Cys. When thethiol-containing conjugate group is an amino acid or a peptide, it mayhave an N-terminal cap, a C-terminal cap, or both. In a preferredembodiment, the thiol-containing conjugate group has both an N-terminalcap and a C-terminal cap. In some embodiments, the thiol-containingconjugating group is itself a peptide comprising the amino acid sequenceof SEQ ID NO:155. In some embodiments, the thiol-containing conjugatinggroup and the peptide are the same (i.e., the conjugate is a dimer).

In some embodiments, the teachings include a conjugate of athiol-containing conjugating group and a peptide comprising the aminoacid sequence(D-Cys)-(X_(aa2))—(X_(aa3))—(X_(aa4))—(X_(aa5))—(X_(aa6))—(X_(aa7)) (SEQID NO:156), wherein (X_(aa2)) is a non-cationic amino acid residue,(X_(aa3)) is any amino acid residue, (X_(aa4)) is any amino acidresidue, (X_(aa5)) is selected from the group consisting of D-Arg,L-Arg, D-Lys and L-Lys, (X_(aa6)) is a non-cationic residue, and(X_(aa7)) is any amino acid residue. The peptide may have an N-terminalcap, a C-terminal cap, or both. In a preferred embodiment, the peptidehas both an N-terminal cap and a C-terminal cap. In a preferredembodiment, the thiol-containing conjugating group is selected from thegroup consisting of D-Cys, L-Cys, a peptide containing D-Cys, and apeptide containing L-Cys. When the thiol-containing conjugate group isan amino acid or a peptide, it may have an N-terminal cap, a C-terminalcap, or both. In a preferred embodiment, the thiol-containing conjugategroup has both an N-terminal cap and a C-terminal cap. In someembodiments, the thiol-containing conjugating group is itself a peptidecomprising the amino acid sequence of SEQ ID NO:156. In someembodiments, the thiol-containing conjugating group and the peptide arethe same (i.e., the conjugate is a dimer).

In some embodiments, the teachings include a conjugate of athiol-containing conjugating group and a peptide comprising the aminoacid sequence(L-Cys)-(X_(aa2))—(X_(aa3))—(X_(aa4))—(X_(aa5))—(X_(aa6))—(X_(aa7)) (SEQID NO:183), wherein (X_(aa2)) is a non-cationic amino acid residue,(X_(aa3)) is any amino acid residue, (X_(aa4)) is any amino acidresidue, (X_(aa5)) is selected from the group consisting of D-Arg,L-Arg, D-Lys and L-Lys, (X_(aa6)) is a non-cationic residue, and(X_(aa7)) is any amino acid residue. The peptide may have an N-terminalcap, a C-terminal cap, or both. In a preferred embodiment, the peptidehas both an N-terminal cap and a C-terminal cap. In a preferredembodiment, the thiol-containing conjugating group is selected from thegroup consisting of D-Cys, L-Cys, a peptide containing D-Cys, and apeptide containing L-Cys. When the thiol-containing conjugate group isan amino acid or a peptide, it may have an N-terminal cap, a C-terminalcap, or both. In a preferred embodiment, the thiol-containing conjugategroup has both an N-terminal cap and a C-terminal cap. In someembodiments, the thiol-containing conjugating group is itself a peptidecomprising the amino acid sequence of SEQ ID NO:183. In someembodiments, the thiol-containing conjugating group and the peptide arethe same (i.e., the conjugate is a dimer).

In some embodiments, the teachings include a conjugate of athiol-containing conjugating group and a peptide comprising the aminoacid sequence(D-Cys)-(D-Ala)-(X_(aa3))—(X_(aa4))-(D-Arg)-(D-Ala)-(X_(aa7)) (SEQ IDNO:161), wherein (X_(aa3)) is any amino acid residue, (X_(aa4)) is anyamino acid residue, and (X_(aa7)) is any amino acid residue. The peptidemay have an N-terminal cap, a C-terminal cap, or both. In a preferredembodiment, the peptide has both an N-terminal cap and a C-terminal cap.In a preferred embodiment, the thiol-containing conjugating group isselected from the group consisting of D-Cys, L-Cys, a peptide containingD-Cys, and a peptide containing L-Cys. When the thiol-containingconjugate group is an amino acid or a peptide, it may have an N-terminalcap, a C-terminal cap, or both. In a preferred embodiment, thethiol-containing conjugate group has both an N-terminal cap and aC-terminal cap. In some embodiments, the thiol-containing conjugatinggroup is itself a peptide comprising the amino acid sequence of SEQ IDNO:161. In some embodiments, the thiol-containing conjugating group andthe peptide are the same (i.e., the conjugate is a dimer).

III. Methods of Use

In one aspect, methods to prevent, treat or amelioratehyperparathyroidism, bone disease and/or other hypercalcemic disordersby administering the compounds described herein are contemplated. Asillustrated above, the compounds have activity to decrease PTH and/orcalcium levels in a target tissue or tissues, or in a subject. Incertain embodiments, the described compounds are capable of decreasingPTH and/or calcium levels when a therapeutically effective amount of thecompound is administered to a subject in need of such treatment. Themethods of use will now be described with reference to Examples 3 and8-11.

With reference again to Example 3, and as discussed above with respectto Table 1, the series of compounds where a cationic (arginine) residuewas sequentially replaced with a non-cationic residue (alanine) wereadministered to rats. FIG. 5 shows the time profile of each compound'sability to reduce blood PTH and the duration of action of the varyingcompounds. In FIG. 5, the compounds Ac-crrrrrr-NH₂ (SEQ ID NO:6,diamonds), Ac-carrrrr-NH₂ (SEQ ID NO:8, squares) and Ac-crrarrr-NH₂ (SEQID NO:10, x symbols) and Ac-crrrrar-NH₂ (SEQ ID NO:12, circles) werepotent in vivo, as evidenced by the decrease in percent PTH of predosebaseline to essentially zero and provided a duration of potency, wherethe PTH blood concentration remained decreased for at least four hours.The compounds Ac-crarrrr-NH₂ (SEQ ID NO:9, triangles), Ac-crrrarr-NH₂(SEQ ID NO:11, * symbols) and Ac-crrrrra-NH₂ (SEQ ID NO:13, +symbols)decreased percent PTH of baseline for about 2-3 hours, and thereafterthe blood concentration of PTH began to increase. Substitution of thecationic (arginine) residue at subunit positions 5 or 7 ofAc-crrrrrr-NH₂ (SEQ ID NO:6) impacted the duration of PTH loweringactivity.

The profile of PTH reduction for a series of compounds containing doubleamino acid substitutions was also evaluated. Selected compounds setforth in Table 2, above, were administered to normal rats by IV bolus ata dose of 0.5 mg/kg and the reduction in PTH relative to predose PTHblood level was evaluated. Data are shown in FIGS. 6A-6B, where thecompound are identified as follows: Ac-carrrar-NH₂ (SEQ ID NO:26, opendiamonds), Ac-crrarar-NH₂ (SEQ ID NO:25, open squares), Ac-caarrrr-NH₂(SEQ ID NO:22, triangles), Ac-crraarr-NH₂ (SEQ ID NO:17, circles),Ac-c(C)arrrar-NH₂ (SEQ ID NO:3, diamonds FIG. 6B), Ac-c(C)rrarar-NH₂(SEQ ID NO:28, x symbols, FIG. 6B).

Another study was done to further evaluate the potency of the compoundAc-c(C)arrrar-NH₂ (SEQ ID NO: 3). The compound was intravenouslyadministered to normal rats, as detailed in Example 2, at doses of 1mg/kg, 0.5 mg/kg, 0.3 mg/kg, and 0.1 mg/kg. Plasma PTH levels wereassessed prior to dosing and for 4 hours thereafter. FIG. 7 shows theresults, where the PTH blood concentration is shown as percent of thebaseline pre-dose value. A dose-related PTH reduction was observedfollowing a single IV bolus administration with the highest dose of 1mg/kg (diamonds) had the largest reduction in PTH, followed by the 0.5mg/kg (squares), 0.3 mg/kg (triangles), and 0.1 mg/kg (x symbols). Thesaline control is shown by the circles symbols. As seen, the peptidewhen administered at a therapeutically effective dose achieves areduction in PTH of greater than 50% relative to the concentration ofPTH before dosing (“baseline”). Specifically, the peptide whenadministered at doses of greater than 0.1 mg/kg reduced PTHconcentration to less than 90% of the baseline PTH concentration 1 hourafter IV administration. These doses of the peptide identified as SEQ IDNO:3 also achieved an area under the curve (AUC) of less than 50%, theAUC calculated as the sum of the PTH concentration values at the timepoints of 1, 2, 3 and 4 hours, normalized by the AUC for the salinecontrol at the same time points, multiplied by 100.

The same compound was also tested in subjects (rats) with renalinsufficiency. In this study, the 1K1C model of acute renalinsufficiency was used to evaluate a the compound Ac-c(C)arrrar-NH₂ (SEQID NO: 3) to characterize its PTH-lowering activity in a renaldysfunction environment. The model is described in Example 1A. Thecompound was intravenously administered as a bolus to renallycompromised animals (rats) at doses of 3 mg/kg (n=2), 1 mg/kg (n=5), 0.5mg/kg (n=6) and 0.3 mg/kg (n=5). A control group of animals was dosedwith saline. Plasma PTH levels were assessed prior to dosing and forseveral hours thereafter. FIG. 8 shows the results, where the salinetreated animals (squares) had an increased PTH concentration relative tothe starting PTH level. At various doses of SEQ ID NO:3, adose-dependant effect was observed on the duration and extent of PTHreduction. Animals treated with the lowest dose of 0.3 mg/kg (x symbols)exhibiting reduced PTH at the earliest time point and an increase in PTHbetween hours 1-24 after dosing. The dose levels of 3 mg/kg (diamonds),1 mg/kg (triangles) and 0.5 mg/kg (squares) provided a reduced PTH bloodconcentration for more than 15 hours, and for the highest dose, for morethan 24 hours.

In another study to evaluate the effect of substituting cationicsubunits with uncharged subunits, as exemplified by alanine amino acidresidues, in the context of a subject with renal insufficiency, ananalog of Ac-crrrrrr-NH₂ (SEQ ID NO:6) was generated and tested for itsability to lower PTH in 1K1C model animals following a 1 mg/kg singleintravenous administration. In the tested analog Ac-carrrar-NH₂ (SEQ IDNO:26), the cationic subunits at positions X₂ and X₆ of Ac-crrrrrr-NH₂(SEQ ID NO:6) were substituted with uncharged amino acids.

As shown in FIG. 9, Ac-carrrar-NH₂ (SEQ ID NO:26, open squares) showsactivity that is equivalent to Ac-crrrrrr-NH₂ (SEQ ID NO:6, opendiamonds) at the dose tested (1 mg/kg) with similar extended duration ofaction over 24 hours. The analog of Ac-crrrrrr-NH₂ (SEQ ID NO:6) withuncharged subunit substitutions was found to retain activity and may, infact, have in vivo potency and duration of action superior to that ofthe compound identified as SEQ ID NO:6. In the compound, Ac-carrrar-NH₂(SEQ ID NO:26), D-Arg residues at positions X₂ and X₆ were substitutedwith D-Ala residues relative to the compound identified as SEQ ID NO:6.

Significantly, as discussed above, administration of the compoundAc-carrrar-NH₂ (SEQ ID NO:26) was not accompanied with histaminerelease, an undesirable side-effect that is seen with Ac-crrrrrr-NH₂(SEQ ID NO:6) and other similar compounds when administered at higherdoses (>1 mg/kg) by IV bolus. The marked attenuation of the histaminerelease with the compound identified SEQ ID NO:26 increases thetherapeutic margin between the desired PTH-lowering activity and theundesired histamine-inducing activity following administration by IVbolus. Accordingly, in a preferred embodiment, compounds having activityto reduce PTH concentration in vivo in the absence of a histamineresponse are provided. Accordingly, in one embodiment, a compound isprovided that has activity to decrease PTH where the compound whenadministered an a subject, human or otherwise, decreases PTH level tobelow 50% of the pre-dose level within one hour after dosing. In aspecific embodiment, a compound that has significant activity todecrease PTH intends a compound that when administered to a normal ratdecreases PTH level to below 50% of the pre-dose level within one hourafter dosing by IV bolus.

In another study, detailed in Example 8, compounds in the form of aconjugate, where the thiol-containing subunit in position X₁ was linkedthrough a disulfide linkage to an L-Cys residue. These compounds havethe following structures:

   C Ac-C    |    | Ac-carrrar-NH₂ and Ac-carrrar-NH₂ (SEQ ID NO: 3)(SEQ ID NO: 141)

In the notation used herein, the compound that is linked to thethiol-containing moiety in the X₁ subunit is identified parenthetically,where in these exemplary conjugates the compound L-Cys is indicated (C)is linked to the thiol-containing moiety in the X₁ subunit:Ac-c(C)arrrar-NH₂ (SEQ ID NO:3) and Ac-c(Ac-C)arrrar-NH₂ (SEQ IDNO:141). These compounds were administered via IV bolus to animals withacute renal insufficiency (1K1C model) at doses of 0.3 and 0.5 mg/kg,and the results are shown in FIG. 10. The compound Ac-c(C)arrrar-NH₂(SEQ ID NO:3) is represented by squares (0.3 mg/kg, n=5) and * symbols(0.5 mg/kg, n=6) and the compound Ac-c(Ac-C)arrrar-NH₂ (SEQ ID NO:141)by triangles (0.3 mg/kg, n=8) and diamonds (0.5 mg/kg, n=7). This invivo dose response with SEQ ID NO:3 displays a dose-dependent reductionin PTH very similar to Ac-crrrrrr-NH₂ (SEQ ID NO:6).

In some of the in vivo studies described herein, the compounds,including compounds in conjugate form where the thiol in the X₁ subunitis cross-linked via a disulfide bond to another subunit, wereadministered as a 30-minute IV infusion. However, it should be notedthat shorter infusions (e.g., <5 minutes) or delivery by IV bolustypically produce comparable pharmacodynamic reduction of PTH as alonger 30-minute infusion. Subcutaneous bolus administration also provedto be an efficacious route of delivery that generated a smaller initialdrop in PTH but displayed a sustained reduction in PTH similar to theprofile seen by the IV route. As shown in FIG. 11, the compoundAc-crrrrrr-NH₂ (SEQ ID NO:6), was also administered bymicropore-facilitated (e.g., microporation of the stratum corneum)transdermal delivery, and demonstrated a reduction in plasma PTH for theseveral hours it was monitored. The compound Ac-crrrrrr-NH₂ (SEQ IDNO:6), was also administered by the transdermal route aftermicroporation resulting in a reduction in plasma PTH for several hours.Transdermal delivery of Ac-crrrrrr-NH₂ (SEQ ID NO:6) provides anaddition option for clinical delivery of the described compounds.

To evaluate the effect of administration route on the activity ofAc-crrrrrr-NH₂ (SEQ ID NO:6) in the context of a subject with renalinsufficiency, rats in the 1K1C model were given 1 mg/kg of the peptideas either a subcutaneous (SC) bolus or a 30-minute IV infusion. Bothroutes of administration effectively reduced plasma PTH levels for over24 hours. When Ac-crrrrrr-NH₂ (SEQ ID NO:6) was delivered by IVinfusion, PTH levels fell rapidly by 80-90% from baseline. By 16 hoursafter dosing, PTH levels had started to rise although they were stillreduced by ˜80% from baseline. When Ac-crrrrrr-NH₂ (SEQ ID NO:6) wasdelivered by SC bolus, PTH levels exhibited a more moderate initial dropto ˜40% of baseline, but exhibited a similar duration of reduction aswhen the peptide was delivered by the IV route. Twenty-four hours afterdosing, PTH levels in animals dosed by either route had partiallyrebounded although both still displayed reduced PTH levels that were˜40-60% from baseline. The results showed that this route ofadministration provides a similar profile with respect to efficacy andduration of PTH reduction as IV administration, thus providing analternative path for clinical dosing (data not shown).

Accordingly, in a preferred embodiment, a subject having secondaryhyperparathyroidism (SHPT) is treated using the described compounds toreduce plasma PTH levels and/or calcium. Untreated SHPT patients withmoderately severe hyperparathyroidism often have baseline circulatingintact PTH levels >300 μg/ml, and levels that can exceed 600 μg/mL. In apreferred embodiment, the decrease in PTH levels is measured as adecrease in intact PTH below pretreatment baseline levels. In anotherembodiment the desired decrease in PTH is to bring the plasma PTH levelsinto generally recognized guidelines established by the National KidneyFoundation or other experts in the treatment of kidney disorders andrenal insufficiency.

In another aspect, methods for treating hyperparathyroidism,hypercalcemia and/or bone disease are provided, comprising administeringa therapeutically effective amount of a described compound. In anotherembodiment, the subject can be treated with a described compound incombination with one or more other therapeutically effective agents.

In another aspect, the described compound is administered in an amounteffective to reduce PTH or PTH effect. In some embodiments, thereduction in plasma PTH is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25% or 30% belowpretreatment baseline levels for at least 10 hours post administrationof the described compound. In specific embodiments, the reduction inplasma PTH is at least 20% at 10 hours post administration. In preferredembodiments, the reduction in plasma PTH is 15 to 40%, preferably 20 to50%, more preferably 30 to 70% below pretreatment baseline levels for atleast 48 hours post administration of the described compound.

In another aspect, the described compound is administered in an amounteffective to decrease serum calcium or calcium effect. In someembodiments, the reduction in serum calcium is at least 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,20% or 25% below pretreatment levels for at least 10 hours postadministration of the polycationic peptide. In some preferredembodiments, the reduction in serum calcium is at least 5% at 10 hourspost administration. In some preferred embodiments, the reduction isserum calcium is 5 to 10%, preferably 5 to 20% below pretreatment levelsfor at least 48 hours post administration of the described compound.

In another aspect, a method for treating hyperparathyroidism and/orhypercalcemia in a subject in need thereof is provided, comprising:administering a therapeutically effective amount of a describedcompound, whereby PTH and/or calcium is reduced.

Based on the relationship between serum calcium, bone metabolism andPTH, it is thought that the described compounds are beneficial for thetreatment of various forms of bone disease and/or hypercalcemia inaddition to hyperparathyroidism. The described compounds may haveadvantages compared to current therapeutic agents, because they may beadministered parenterally and may not be associated withgastrointestinal adverse effects, are not metabolized by cytochrome P450and may result in more effective reductions in plasma PTH and calcium.

As discussed above, the described methods may be used alone or incombination with one or more other therapeutically effective agents.Such other therapeutically effective agents include, but are not limitedto, treatment with antiresorptive bisphosphonate agents, such asalendronate and risedronate; integrin blockers, such as α_(v)β₃antagonists; conjugated estrogens used in hormone replacement therapy,such as PREMPRO™, PREMARIN™ and ENDOMETRION™; selective estrogenreceptor modulators (SERMs), such as raloxifene, droloxifene, CP-336,156(Pfizer) and lasofoxifene; cathespin K inhibitors; vitamin D therapy;vitamin D analogs, such as ZEMPLAR™ (paricalcitol); CALCIJEX®(calcitriol), HECTOROL® (doxercalciferol), ONE-ALPHA® (alfacalcidol) andthe analogs in development from Cytochroma known as CTA-018, CTAP201 andCTAP101; other calcimimetics such as Sensipar® (cinacalcet); inhibitorsof type II sodium-dependent phosphate transporter family, SLC34(including the two renal isoforms NaPi-Ila and NaPi-IIc, and theintestinal NaPi-IIb transporter); phosphatonins (including FGF-23,sFRP4, MEPE or FGF-7); low dose PTH treatment (with or withoutestrogen); calcitonin; inhibitors of RANK ligand; antibodies againstRANK ligand, osteoprotegrin; adensosine antagonists; and ATP proton pumpinhibitors.

In one embodiment, a described compound is administered at a dosesufficient to decrease both PTH and serum calcium levels. In anotherembodiment, a described compound is administered at a dose sufficient todecrease PTH without significantly affecting serum calcium levels. In afurther embodiment, a described compound is administered at a dosesufficient to increase PTH without significantly affecting serum calciumlevels.

Formulations

A pharmaceutical composition comprising a described compound and atleast one pharmaceutically acceptable excipient or carrier is provided.Methods of preparing such pharmaceutical compositions typically comprisethe step of bringing into association a described compound with acarrier and, optionally, one or more accessory ingredients. Thedescribed compounds and/or pharmaceutical compositions comprising samemay be formulated into pharmaceutically-acceptable dosage forms byconventional methods known to those of skill in the art. Typically,formulations are prepared by uniformly and intimately bringing intoassociation a described compound with liquid carriers, or finely dividedsolid carriers, or both, and then, if necessary, shaping the product.

Pharmaceutical compositions of the present invention suitable forparenteral administration comprise one or more described compounds incombination with one or more pharmaceutically-acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containsugars, alcohols, amino acids, antioxidants, buffers, bacteriostats,solutes which render the formulation isotonic with the blood of theintended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These pharmaceutical compositions may also contain adjuvants such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms upon the described compoundsmay be ensured by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol sorbic acid, and thelike. It may also be desirable to include agents to control tonicity,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

For example, a described compound may be delivered to a human in a formof solution that is made by reconstituting a solid form of the drug withliquid. This solution may be further diluted with infusion fluid such aswater for injection, 0.9% sodium chloride injection, 5% dextroseinjection and lactated ringer's injection. It is preferred that thereconstituted and diluted solutions be used within 4-6 hours fordelivery of maximum potency. Alternatively, a described compound may bedelivered to a human in a form of tablet or capsule.

Injectable depot forms are made by forming microencapsulated matrices ofthe described compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the described compounds are administered as pharmaceuticals, tohumans and animals, they can be given alone or as a pharmaceuticalcomposition containing, for example, 0.1 to 99% (more preferably, 10 to30%) of active ingredient in combination with a pharmaceuticallyacceptable carrier. In other embodiments, the pharmaceutical compositionmay contain 0.2-25%, preferably 0.5-5% or 0.5-2%, of active ingredient.These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including, e.g.,subcutaneous injection, subcutaneous depot, intravenous injection,intravenous or subcutaneous infusion. These compounds may beadministered rapidly (within <1 minute) as a bolus or more slowly overan extended period of time (over several minutes, hours or days). Thesecompounds may be delivered daily or over multiple days, continuously orintermittently. In one embodiment, the compounds may be administeredtransdermally (e.g., using a patch, microneedles, micropores, ointment,microjet or nanojet).

Regardless of the route of administration selected, the describedcompounds, which may be used in a suitable hydrated form, and/or thepharmaceutical compositions, are formulated intopharmaceutically-acceptable dosage forms by conventional methods knownto those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient which is effective to achieve the desired therapeuticresponse for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular described compound employed, orthe ester, salt or amide thereof, the route of administration, the timeof administration, the rate of excretion or metabolism of the particularcompound being employed, the rate and extent of absorption, the durationof the treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the described compounds employed in the pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved.

In general, a suitable daily dose of a described compound will be thatamount of the compound which is the lowest dose effective to produce atherapeutic effect. Such an effective dose will generally depend uponthe factors described above. Generally, intravenous, intramuscular,transdermal, intracerebroventricular and subcutaneous doses of thedescribed compounds for a patient, when used for the indicated effects,will range from about 1 μg to about 5 mg per kilogram of body weight perhour. In other embodiments, the dose will range from about 5 μg to about2.5 mg per kilogram of body weight per hour. In further embodiments, thedose will range from about 5 μg to about 1 mg per kilogram of bodyweight per hour.

If desired, the effective daily dose of a described compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms. In one embodiment, the describedcompound is administered as one dose per day. In further embodiments,the compound is administered continuously, as through intravenous orother routes. In other embodiments, the compound is administered lessfrequently than daily, such as every 2-3 days, in conjunction withdialysis treatment, weekly or less frequently.

The subject receiving this treatment is any animal in need, includingprimates, in particular humans, and other mammals such as equines,cattle, swine and sheep; and poultry and pets in general.

The described compounds may be administered as such or in admixtureswith pharmaceutically acceptable carriers and can also be administeredin conjunction with antimicrobial agents such as penicillins,cephalosporins, aminoglycosides and glycopeptides. Conjunctive therapythus includes sequential, simultaneous and separate administration ofthe active compound in a way that the therapeutical effects of the firstadministered one is not entirely disappeared when the subsequent isadministered.

Routes of Administration for Disclosed Compounds

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration. As used herein, theterm “route” of administration is intended to include, but is notlimited to subcutaneous injection, subcutaneous depot, intravenousinjection, intravenous or subcutaneous infusion, intraocular injection,intradermal injection, intramuscular injection, intraperitonealinjection, intratracheal administration, intraadiposal administration,intraarticular administration, intrathecal administration, epiduraladministration, inhalation, intranasal administration, sublingualadministration, buccal administration, rectal administration, vaginaladministration, intracisternal administration and topicaladministration, transdermal administration, or administration via localdelivery (for example by catheter or stent).

Transdermal drug delivery to the body is a desirable and convenientmethod for systemic delivery of biologically active substances to asubject, and in particular for delivery of substances that have poororal bioavailability, such as proteins and peptides. The transdermalroute of delivery has been particularly successful with small (e.g.,less than about 1,000 Daltons) lipophilic compounds, such as scopolamineand nicotine, that can penetrate the stratum corneum outer layer of theskin, which serves as an effective barrier to entry of substances intothe body. Below the stratum corneum is the viable epidermis, whichcontains no blood vessels, but has some nerves. Deeper still is thedermis, which contains blood vessels, lymphatics and nerves. Drugs thatcross the stratum corneum barrier can generally diffuse to thecapillaries in the dermis for absorption and systemic distribution.

Technological advances in transdermal delivery have focused onaddressing the need in the art to deliver hydrophilic, high molecularweight compounds, such as proteins and peptides, across the skin. Oneapproach involves disruption of the stratum corneum using chemical orphysical methods to reduce the barrier posed by the stratum corneum.Skin microporation technology, which involves the creation of microndimension transport pathways (micropores) in the skin (in particular,the micropores in the stratum corneum) using a minimally invasivetechnique, is a more recent approach. Techniques to create micropores inthe skin (stratum corneum) include thermal microporation or ablation,microneedle arrays, phonophoresis, laser ablation and radiofrequencyablation (Prausnitz and Langer (2008) Nat. Biotechnology 11:1261-68;Arora et al., Int. J. Pharmaceutics, 364:227 (2008); Nanda et al.,Current Drug Delivery, 3:233 (2006); Meidan et al. American J.Therapeutics, 11:312 (2004)).

As noted above, PTH secretion is regulated by the CaSR which isexpressed on the cell surface of parathyroid cells. Thus, in order toactivate the CaSR, the agent or compound must be delivered to theparathyroid cell. Transdermal delivery of calcimimetic agents mustachieve delivery across the stratum corneum and provide systemicexposure to reach the parathyroid cell. To date, the art has notdemonstrated whether a calcimimetic compound can be deliveredtransdermally in an amount sufficient for therapeutic benefit and inparticular in an amount sufficient for decreasing PTH and/or thetreatment, attenuation, lessening and/or relief hypercalcemia.

In addition to calcimimetics, 1,25-(OH)₂ vitamin D₃ analogs are the mostcommonly used treatments for patients with hyperparathyroidismassociated with chronic kidney disease and end stage renal disease.Vitamin D analogs act by facilitating intestinal absorption of dietarycalcium, and reduce PTH levels by inhibiting PTH synthesis andsecretion. While intravenous and oral delivery of vitamin D has beenused therapeutically, to date, the art has not demonstrated whethervitamin D analogs, such as ZEMPLAR™ (paricalcitol), CALCIJEX®(calcitriol), ONE-ALPHA® (alfacalcidol) and HECTOROL® (doxercalciferol)can be delivered transdermally in an amount sufficient for therapeuticbenefit and in particular in an amount sufficient for decreasingparathyroid hormone (PTH). In addition, the art has not demonstratedwhether the co-administration by transdermal delivery of a calcimimeticagent in combination with a vitamin D analog (either as separateformulations or as a co-formulation) in amounts sufficient fortherapeutic benefit, and in particular in amounts sufficient fordecreasing PTH and provide effective treatment for patients sufferingfrom hyperparathyroidism.

The calcimimetic agents may be administered across the stratum corneum,and/or other layers of the epidermis, for local or systemic delivery,for decreasing parathyroid hormone (PTH) and/or treating hypercalcemia.In one embodiment, the calcimimetic agent is delivered viamicroporation. Any one of a number of techniques for microporation iscontemplated, and several are briefly described.

Microporation can be achieved by mechanical means and/or externaldriving forces, to breach the stratum corneum to deliver thecalcimimetic agents described herein through the surface of the skin andinto the underlying skin layers and/or the bloodstream.

In a first embodiment, the microporation technique is ablation of thestratum corneum in a specific region of the skin using a pulsed laserlight of wavelength, pulse length, pulse energy, pulse number, and pulserepetition rate sufficient to ablate the stratum corneum withoutsignificantly damaging the underlying epidermis. The calcimimetic agentis then applied to the region of ablation. Another laser ablationmicroporation technique, referred to as laser-induced stress waves(LISW), involves broadband, unipolar and compressible waves generated byhigh-power pulsed lasers. The LISWs interact with tissues to disrupt thelipids in the stratum corneum, creating intercellular channelstransiently within the stratum corneum. These channel, or micropores, inthe stratum corneum permit entry of the calcimimetic agent.

Sonophoresis or phonophoresis is another microporation technique thatuses ultrasound energy. Ultrasound is a sound wave possessingfrequencies above 20 KHz. Ultrasound can be applied either continuouslyor pulsed, and applied at various frequency and intensity ranges (Nandaet al., Current Drug Delivery, 3:233 (2006)).

Another microporation technique involves the use of a microneedle array.The array of microneedles when applied to a skin region on a subjectpierce the stratum corneum and do not penetrate to a depth thatsignificantly stimulates nerves or punctures capillaries. The patient,thus, feels no or minimal discomfort or pain upon application of themicroneedle array for generation of micropores through which thecalcimimetic agent is delivered.

Microneedle arrays comprised of hollow or solid microneedles arecontemplated, where the calcimimetic agent can be coated on the externalsurface of the needles or dispensed from the interior of hollow needles.Examples of microneedle arrays are described, for example, in Nanda etal., Current Drug Delivery, 3:233 (2006) and Meidan et al. American J.Therapeutics, 11:312 (2004). First generation microneedle arrays werecomprised of solid, silicon microneedles that were externally coatedwith a therapeutic agent. When the microarray of needles was pressedagainst the skin and removed after about 10 seconds, the permeation ofthe agent on the needles into the body was readily achieved. Secondgeneration microneedle arrays were comprised of microneedles of solid orhollow silicon, polycarbonate, titanium or other suitable polymer andcoated or filled with a solution of the therapeutic compound. Newergenerations of microneedle arrays are prepared from biodegradablepolymers, where the tips of the needles coated with a therapeutic agentremain in the stratum corneum and slowly dissolve.

The microneedles can be constructed from a variety of materials,including metals, ceramics, semiconductors, organics, polymers, andcomposites. Exemplary materials of construction include pharmaceuticalgrade stainless steel, gold, titanium, nickel, iron, tin, chromium,copper, palladium, platinum, alloys of these or other metals, silicon,silicon dioxide, and polymers. Representative biodegradable polymersinclude polymers of hydroxy acids such as lactic acid and glycolic acidpolylactide, polyglycolide, polylactide-co-glycolide, and copolymerswith poly(ethylene glycol), polyanhydrides, poly(ortho)esters,polyurethanes, poly(butyric acid), poly(valeric acid), andpoly(lactide-co-caprolactone). Representative non-biodegradable polymersinclude polycarbonate, polyester, and polyacrylamides.

The microneedles can have straight or tapered shafts. In one embodiment,the diameter of the microneedle is greatest at the base end of themicroneedle and tapers to a point at the end distal the base. Themicroneedle can also be fabricated to have a shaft that includes both astraight (untapered) portion and a tapered portion. The needles may alsonot have a tapered end at all, i.e. they may simply be cylinders withblunt or flat tips. A hollow microneedle that has a substantiallyuniform diameter, but which does not taper to a point, is referred toherein as a “microtube.” As used herein, the term “microneedle” includesboth microtubes and tapered needles unless otherwise indicated.

Electroporation is another technique for creating micropores in theskin. This approach uses the application of microsecond or millisecondlong high-voltage electrical pulses to created transient, permeablepores within the stratum corneum.

Other microporation techniques include use of radio waves to createmicrochannels in the skin. Thermal ablation is yet another approach toachieve delivery of larger molecular weight compounds transdermally.

Applicants have discovered that low doses of calcimimetic agents may betherapeutically administered over an extended period of time to treatSHPT. This markedly differs from current dose requirements of othercalcimimetics (e.g., cinacalcet hydrochloride).

Transdermal delivery of the compounds described herein was demonstratedin the studies described in Examples 9-10. In a first study, thecompound Ac-crrrrrr-NH₂ (SEQ ID NO:6) was administered transdermally torats in which a small area of the skin was microporated by 5 passes of a1.0 mm derma roller under moderate pressure. A solution of eitherAc-crrrrrr-NH₂ (SEQ ID NO: 6) or saline was placed on the microporatedarea of skin. Blood draws were taken over a 4 hour period and plasma wasanalyzed for PTH levels by ELISA. The results are shown in FIG. 11,where the plasma PTH is shown as a percent of pre-dose baseline for thesaline treated animal (diamonds) and the two animals treated with thetest compound (squares, triangles). These data indicate that thecompound Ac-crrrrrr-NH₂ (SEQ ID NO: 6) can be delivered systemically insufficient quantities by transdermal route (in this case,micropore-facilitated transdermal delivery) using a derma roller toeffectively and significantly reduce PTH levels from baseline for the ˜4hours that were studies. It should be noted that Ac-crrrrrr-NH₂ (SEQ IDNO: 6) has been shown to effectively reduce PTH levels from baseline inthe 1K1C rat model of acute renal insufficiency when administered byshort IV infusion as well as in normal rats (data not shown).

In another study, described in Example 10, the compoundAc-c(C)arrrar-NH₂ (SEQ ID NO:3) was administered micropore-facilitatedtransdermal delivery to normal rats using a transdermal patch. Atransdermal patch system containing 10% solution (by weight) ofAc-c(C)arrrar-NH₂ (SEQ ID NO:3) in saline was placed over themicroporated area and left in place for ˜30 hours. Blood draws weretaken from the rats periodically over the 30 hours and plasma sampleswere analyzed for PTH levels by ELISA. The results are shown in FIG. 12.Surprisingly, these data demonstrate that micropore-facilitatedtransdermal delivery can achieve sufficient sustained delivery ofAc-c(C)arrrar-NH₂ (SEQ ID NO:3) to produced a significant and extendedreduction in PTH for >30 hours in rats with normal renal function. Thesedata demonstrate that microporation facilitated transdermal delivery ofthe conjugate Ac-c(C)arrrar-NH₂ (SEQ ID NO:3) using a patch can achievesufficient blood exposure of the peptide over the course of treatment toproduce a significant and sustained reduction in PTH from baselinefor >30 hrs. These data demonstrate that transdermal patch delivery on adaily basis or longer would enable treatment of both dialysis andnon-hemodialysis patients in need of treatment. For example, CKD (stage4), primary hyperparathyroidism and secondary hyperparathyroidism (SHPT)in renal transplant patients who are not typically treated with IVdrugs, but could be readily treated by a daily transdermal patch viamicroporation facilitated transdermal delivery.

Another study was conducted to further evaluate the route ofadministration of the compounds. As described in Example 11, thecompound Ac-c(C)arrrar-NH₂ (SEQ ID NO:3) was administered by very lowdose IV infusion to normal rats and to rats with renal insufficiency toidentify the lowest dose needed to be administered by infusion,transdermal patch system or other sustained delivery means to achievesignificant PTH reduction. Healthy rats were intravenously infused forsix hours with very low doses (1 μg/kg/hr, 3 μg/kg/hr, and 10 μg/kg/hr)of Ac-c(C)arrrar-NH₂ (SEQ ID NO:3). Blood samples were taken prior todosing (pre) and at 2 hours, 4 hours, 6 hours (just prior to the end ofinfusion; EOI) and 8 hours (2 hrs post EOI) after the start of infusionand plasma was analyzed for PTH levels by ELISA. Surprisingly, the datashown in FIG. 13 demonstrate that infusion of very low doses ofAc-c(C)arrrar-NH₂ (SEQ ID NO:3) (1 μg/kg/hr (squares), 3 μg/kg/hr(circles), and 10 μg/kg/hr (triangles)) for 6 hours are effective toproduce significant reduction in PTH from baseline over the course ofinfusion. These data indicate that low doses delivered continuouslycould be as effective as (or even more effective than) much larger dosesdelivery as a single bolus.

The PTH lowering effect was further evaluated in the rat 1K1C model ofacute renal insufficiency. 1K1C model rats were intravenously infusedwith low doses of Ac-c(C)arrrar-NH₂ (SEQ ID NO:3) (30 μg/kg/hr and 100μg/kg/hr) for 6 hours. Blood samples were taken prior to dosing (Pre),and at 2 hours, 4 hours, 6 hours (just prior to the end of infusion;EOI), 8 hours (2 hrs post EOI) and 24 hours after the start of infusionand plasma was analyzed for PTH levels by ELISA. The data shown in FIG.14A demonstrate that IV infusion of low doses of Ac-c(C)arrrar-NH₂ (SEQID NO:3) significantly reduce PTH from baseline levels in the 1K1Cmodel, a model of renal insufficiency where baseline PTH levels can beseen to be from 400 to >1100 μg/mL. Surprisingly, 6 hours of low dose IVinfusion (diamonds, 30 μg/kg/hr and squares, 100 μg/kg/hr) ofAc-c(C)arrrar-NH₂ (SEQ ID NO:3) were able to reduce PTH from baselinefor ˜24 hours. Consistent with this dramatic PTH reduction in the 1K1Crat model, FIG. 14B shows a bar graph plotting serum calcium data inthis acute renal insufficiency, and show a corresponding reduction inserum calcium following low dose IV infusion of Ac-c(C)arrrar-NH₂ (SEQID NO:3). These data demonstrate that Ac-c(C)arrrar-NH₂ (SEQ ID NO:3) isa very potent calcimimetic compound that is able to reduce PTH andcalcium following infusion or delivery of low doses (for example bytransdermal delivery) over the course of ˜24 hours. These data furthersupport the conclusion that low dose sustained delivery of acalcimimetic agent by IV infusion or by micropore-facilitatedtransdermal delivery could be an effective treatment for patients on adaily or less frequent basis.

Combination Therapy

As described above, the methods of use may be used alone or incombination with other approaches for the treatment of hypercalcemiaand/or bone disease. Such other approaches include, but are not limitedto, treatment with agents such as bisphosphonate agents, integrinblockers, hormone replacement therapy, selective estrogen receptormodulators, cathepsin K inhibitors, vitamin D therapy, vitamin Danalogs, such as ZEMPLAR™ (paricalcitol), CALCIJEX® (calcitriol),ONE-ALPHA® (alfacalcidol) and HECTOROL® (doxercalciferol),anti-inflammatory agents, low dose PTH therapy (with or withoutestrogen), calcimimetics, phosphate binders, calcitonin, inhibitors ofRANK ligand, antibodies against RANK ligand, osteoprotegrin, adensosineantagonists and ATP proton pump inhibitors.

In one embodiment, a combination therapy uses vitamin D or a vitamin Danalog in combination with a calcimimetic agent. Vitamin D aids in theabsorption of calcium and functions to maintain normal blood levels ofcalcium and phosphorous. PTH works to enhance calcium absorption in theintestine by increasing the production of 1,25-(OH)₂ vitamin D, theactive form of vitamin D. PTH also stimulates phosphorus excretion fromthe kidney, and increases release from bone.

As discussed above, secondary hyperparathyroidism is characterized by anelevation in parathyroid hormone (PTH) associated with inadequate levelsof active vitamin D hormone. Vitamin D or a vitamin D analog may be usedto reduce elevated PTH levels in treatment of secondaryhyperparathryoidism. In one embodiment, the invention includes apharmaceutical composition comprising a calcimimetic agent and a vitaminD analog.

In one embodiment, the invention includes a pharmaceutical compositioncomprising a calcimimetic agent and ZEMPLAR™ (paricalcitol).Paricalcitol is a synthetic analog of calcitriol, the metabolicallyactive form of vitamin D. The recommended initial dose of Zemplar isbased on baseline intact parathyroid hormone (iPTH) levels. If thebaseline iPTH level is less than or equal to 500 μg/mL, the daily doseis 1 μg and the “three times a week” dose (to be administered not morethan every other day) is 2 μg. If the baseline iPTH is greater than 500μg/mL, the daily dose is 2 μg, and the “three times a week” does (to beadministered not more than every other day) is 4 μg. Thereafter, dosingmust be individualized and based on serum plasma iPTH levels, withmonitoring of serum calcium and serum phosphorus. Paricalcitol isdescribed in U.S. Pat. No. 5,246,925 and U.S. Pat. No. 5,587,497.

In another embodiment, the invention includes a pharmaceuticalcomposition comprising a calcimimetic agent and CALCIJEX® (calcitriol).Calcitriol is the metabolically active form of vitamin D. Therecommended initial dosage for CALCIJEX® (oral) is 0.25 p/day. Thisamount may be increased by 0.25 μg/day at 4- to 8-wk intervals. Normalor only slightly reduced calcium levels may respond to dosages of 0.25μg every other day. For patients on dialysis, the recommended initialdose for CALCIJEX® (IV) is 0.02 μg/kg (1 to 2 μg) 3 times/week, everyother day. This amount may be increased by 0.5 to 1 μg, every 2 to 4 wk.Calcitriol is described in U.S. Pat. No. 6,051,567 and U.S. Pat. No.6,265,392 and U.S. Pat. No. 6,274,169.

In one embodiment, a pharmaceutical composition comprising acalcimimetic agent and HECTOROL® (doxercalciferol) is provided.Doxercalciferol is a synthetic analog of vitamin D that undergoesmetabolic activiation in vivo to form 1 α,25-dihydroxyvitamin D₂, anaturally occurring, biologically active form of vitamin D. Therecommended initial dose of HECTOROL® is 10 μg administered three timesweekly at dialysis (approximately every other day). The initial doseshould be adjusted, as needed, in order to lower blood iPTH into therange of 150 to 300 μg/mL. The dose may be increased at 8-week intervalsby 2.5 μg if iPTH is not lowered by 50% and fails to reach target range.The maximum recommended dose of HECTOROL is 20 μg administered threetimes a week at dialysis for a total of 60 μg per week. Doxercalciferolis described in U.S. Pat. No. 5,602,116 and U.S. Pat. No. 5,861,386 andU.S. Pat. No. 5,869,473 and U.S. Pat. No. 6,903,083.

The particular combination of therapies (therapeutics or procedures) toemploy in a combination regimen will take into account compatibility ofthe desired therapeutics and/or procedures and the desired therapeuticeffect to be achieved. It will also be appreciated that the therapiesemployed may achieve a desired effect for the same disorder (forexample, an inventive compound may be administered concurrently withanother agent used to treat the same disorder), or they may achievedifferent effects (e.g., control of any adverse effects). As usedherein, additional therapeutic agents that are normally administered totreat or prevent a particular disease, or condition, are known as“appropriate for the disease, or condition, being treated”.

A combination treatment of the present invention as defined herein maybe achieved by way of the simultaneous, sequential or separateadministration of the individual components of said treatment.

The compounds or pharmaceutically acceptable compositions thereof mayalso be incorporated into compositions for coating implantable medicaldevices, bio-erodible polymers, implantable pump, and suppositories.Accordingly, in another aspect, a composition for coating an implantabledevice comprising a described compound as described generally above iscontemplated, and a carrier suitable for coating the implantable device.In still another aspect, included is an implantable device coated with acomposition comprising a compound as described generally above, and acarrier suitable for coating said implantable device.

Suitable coatings and the general preparation of coated implantabledevices are described in U.S. Pat. Nos. 6,099,562; 5,886,026; and5,304,121. The coatings are typically biocompatible polymeric materialssuch as a hydrogel polymer, polymethyldisiloxane, polycaprolactone,polyethylene glycol, polylactic acid, ethylene vinyl acetate, andmixtures thereof. The coatings may optionally be further covered by asuitable topcoat of fluorosilicone, polysaccarides, polyethylene glycol,phospholipids or combinations thereof to impart controlled releasecharacteristics in the composition.

Potential Clinical Markers for Determining Treatment Efficacy

Determination of the effectiveness of a described method of treatmentmay be determined by a variety of methods.

Normal levels of serum calcium are in the range of 8.8 mg/dL to 10.4mg/dL (2.2 mmol/L to 2.6 mmol/L). In certain cases, the efficacy oftreatment may be determined by measurement of serum and urinary markersrelated to calcium, including but not limited to, total and ionizedserum calcium, albumin, plasma PTH, PTHrP, phosphate, vitamin D, andmagnesium.

In other cases, efficacy may be determined by measurement of bonemineral density (BMD), or by measurement of biochemical markers for boneformation and/or bone resorption in serum or urine. Potential boneformation markers include, but are not limited to, total alkalinephosphatase, bone alkaline phosphatase, osteocalcin, under-carboxylatedosteocalcin, C-terminal procollagen type I propeptide, and N-terminalprocollagen type I propeptide. Potential bone resorption markersinclude, but are not limited, hydroxyproline, hydroxylysine,glycosyl-galactosyl hydroxylysine, galactosyl hydroxylysine,pyridinoline, deoxypyridinoline, N-terminal crosslinking telopeptide oftype I collagen, C-terminal crosslinking telopeptide of type I collagen,C-terminal crosslinking telopeptide of type I collagen generated byMMPs, bone sialoprotein, acid phosphatase and tartrate-resistant acidphosphatase.

In other cases, efficacy may be determined by the percent reduction inPTH relative to a pre-dosing (baseline) level and/or by achieving adesirable PTH level as generally accepted as being beneficial topatients (for example, guidelines established by the National KidneyFoundation). Still in other cases, efficacy may be determined bymeasurement of the reduction in parathyroid gland hyperplasia associatedwith a hyperparathyroidism disease.

It is expected that when a described method of treatment is administeredto a subject in need thereof, the method of treatment will produce aneffect, as measured by, for example, one or more of: total serumcalcium, ionized serum calcium, total blood calcium, ionized bloodcalcium, albumin, plasma PTH, blood PTH, PTHrP, phosphate, vitamin D,magnesium, bone mineral density (BMD), total alkaline phosphatase, bonealkaline phosphatase, osteocalcin, under carboxylated osteocalcin,C-terminal procollagen type I propeptide, N-terminal procollagen type Ipropeptide, hydroxyproline, hydroxylysine, glycosyl-galactosylhydroxylysine, galactosyl hydroxylysine, pyridinoline,deoxypyridinoline, N-terminal crosslinking telopeptide of type Icollagen, C-terminal crosslinking telopeptide of type I collagen,C-terminal crosslinking telopeptide of type I collagen generated byMMPs, bone sialoprotein, acid phosphatase and tartrate-resistant acidphosphatase. Effects include prophylactic treatment as well as treatmentof existing disease.

A biologically effective molecule may be operably linked to a describedpeptide with a covalent bond or a non-covalent interaction. In specificembodiments, the operably linked biologically effective molecules canalter the pharmacokinetics of the described compounds by virtue ofconferring properties to the compound as part of a linked molecule. Someof the properties that the biologically effective molecules can conferon the described compounds include, but are not limited to: delivery ofa compound to a discrete location within the body; concentrating theactivity of a compound at a desired location in the body and reducingits effects elsewhere; reducing side effects of treatment with acompound; changing the permeability of a compound; changing thebioavailability or the rate of delivery to the body of a compound;changing the length of the effect of treatment with a compound; alteringthe in vitro chemical stability of the compound; altering the in vivostability of the compound, half-life, clearance, absorption,distribution and/or excretion; altering the rate of the onset and thedecay of the effects of a compound; providing a permissive action byallowing a compound to have an effect.

In a further aspect, the described compound may be conjugated topolyethylene glycol (PEG). The selected PEG may be of any convenientmolecular weight, and may be linear or branched, and may be optionallyconjugated through a linker. The average molecular weight of PEG willpreferably range from about 2 kiloDalton (kDa) to about 100 kDa, morepreferably from about 5 kDa to about 40 kDa. Alternatively, the PEGmoiety used can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 kDa.

The described compounds may be conjugated to PEG through a suitableamino acid residue located at any position on the compounds. Thedescribed compounds may optionally contain an additional amino acidresidue to which PEG is conjugated, including for example, an additionalamine-containing residue, such as lysine.

PEGylated peptides are known in the art to increase serum half-life ofconjugated peptide. A variety of methods are known in the art for theformation of PEGylated peptides. For example, the PEG moiety can belinked to the amino terminus, the carboxy terminus or through a sidechain of the claimed peptide, optionally through the presence of alinking group. In other embodiments, the PEG moiety may be linked to thesulfur of a thiol-containing amino acid, such as cysteine, or may becoupled to the sidechain of an amine-containing amino acid, such aslysine.

The PEG groups will generally be attached to the described compound byacylation or alkylation through a reactive group on the PEG moiety(e.g., an aldehyde, amine, oxime, hydrazine thiol, ester, or carboxylicacid group) to a reactive group on the described compound (e.g., analdehyde, amine, oxime, hydrazine, ester, acid or thiol group), whichmay be located at the amino terminus, carboxy terminus, or a sidechainposition of the described compound. One approach for preparation ofPEGylation of synthetic peptides consists of combining through aconjugate linkage in solution, a peptide and a PEG moiety, each bearinga functional group that is mutually reactive towards the other. Peptidescan be easily prepared using conventional solution or solid phasesynthesis techniques. Conjugation of the peptide and PEG is typicallydone in aqueous phase and may be monitored by reverse phase HPLC. ThePEGylated peptides can be readily purified and characterized, usingstandard techniques known to one of skill in the art.

One or more individual subunits of the described compounds may also bemodified with various derivatizing agents known to react with specificside chains or terminal residues. For example, lysinyl residues andamino terminal residues may be reacted with succinic anhydride or othersimilar carboxylic acid anhydrides which reverses the charge on thelysinyl or amino residue. Other suitable reagents include, e.g.,imidoesters such as methyl picolinimidate; pyridoxal; pyridoxalphosphate; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4,-pentanedione; and transaminase-catalyzed reactionwith glyoxalate. Arginyl residues may be modified by reaction withconventional agents such as phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin.

In addition, the described compounds may be modified to includenon-cationic residues that provide immunogenic residues useful for thedevelopment of antibodies for bioanalytical ELISA measurements, as wellas to evaluate immunogenicity. For example, the described compounds maybe modified by incorporation of tyrosine and/or glycine residues.Specific modifications of tyrosyl residues are of particular interestfor introducing spectral labels into tyrosyl residues. Non-limitingexamples include reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidazole andtetranitromethane are used to form O-acetyl tyrosyl and 3-nitroderivatives, respectively.

Kits Comprising the Disclosed Compounds

The invention also provides kits for carrying out the therapeuticregimens of the invention. Such kits comprise therapeutically effectiveamounts of the described compounds having activity as a CaSR modulator,in pharmaceutically acceptable form, alone or in combination with otheragents, in pharmaceutically acceptable form. Preferred pharmaceuticalforms include the described compounds in combination with sterilesaline, dextrose solution, buffered solution, sterile water, or otherpharmaceutically acceptable sterile fluid. Alternatively the compositionmay include an antimicrobial or bacteriostatic agent. Alternatively, thecomposition may be lyophilized or desiccated. In this instance, the kitmay further comprise a pharmaceutically acceptable solution, preferablysterile, to form a solution for injection purposes. In anotherembodiment, the kit may further comprise a needle or syringe, preferablypackaged in sterile form, for injecting the composition. In otherembodiments, the kit further comprises an instruction means foradministering the composition to a subject. The instruction means can bea written insert, an audiotape, an audiovisual tape, or any other meansof instructing the administration of the composition to a subject.

In one embodiment, the kit comprises (i) a first container containing adescribed compound having activity as a CaSR modulator; and (ii)instruction means for use. In another embodiment the kit comprises (i) afirst container containing a compound as described herein, and (ii) asecond container containing a pharmaceutically acceptable vehicle fordilution or reconstitution.

In another embodiment, the kit comprises (i) a first containercontaining a described compound having activity as a CaSR modulator;(ii) a second container containing an anticalcemic agent; and (iii)instruction means for use.

In one embodiment, the anticalcemic agent is and agent selected from thegroup consisting of bisphosphonate agents, hormone replacementtherapeutic agents, vitamin D therapy, vitamin D analogs, such asZEMPLAR™ (paricalcitol); CALCIJEX® (calcitriol), ONE-ALPHA®(alfacalcidol) and HECTOROL® (doxercalciferol), low dose PTH (with orwithout estrogen), and calcitonin.

In related aspects, the invention provides articles of manufacture thatcomprise the contents of the kits described above. For instance, theinvention provides an article of manufacture comprising an effectiveamount of a described peptide, alone or in combination with otheragents, and instruction means indicating use for treating diseasesdescribed herein.

EXAMPLES

The following examples are offered to illustrate but not to limit thecompounds and methods described herein. Various modifications may bemade by the skilled person without departing from the true spirit andscope of the subject matter described herein.

Example 1 Cationic Compounds with PTH Lowering Activity

Model of Renal Insufficiency

A rat model of acute renal insufficiency (also referred to as the 1K1Cmodel) was developed to simulate the pathology of SHPT associated withend stage renal disease. The model exhibits pathological characteristicsof hyper parathyroidsm associated with a lack of renal function,specifically the significant elevation of plasma PTH and reduction inserum calcium. The development of this model allowed for the furthercharacterization of described compounds in the context of a subject withrenal dysfunction and elevated PTH. Typical baseline PTH levels in thismodel averaged ˜450 μg/mL.

The 1K1C model of acute renal insufficiency involves the removal of onekidney followed by exposure of the remaining kidney to 45 minutes ofischemia and 48 hours of reperfusion. The ensuing ischemia/reperfusion(I/R) damage to the remaining kidney results in significant necrosis andrenal failure. Serum creatinine levels were elevated for over 24-48hours following I/R insult (data not shown). Also due to the resultingrenal dysfunction, total PTH levels are dramatically increased from thepre-I/R injury levels of ˜100 μg/mL. By 48 hours post-I/R, plasma PTHlevels were elevated to ˜450 μg/mL (˜5 fold increase) and in someinstances reach as high as ˜1200 μg/mL. This reproducible increase inserum creatinine and PTH provided a robust model that mimics thephysiology seen ESRD patients.

Cinacalcet hydrochloride (SENSIPAR®), an approved calcimimetic agentthat is used to lower PTH for the treatment of SHPT, was tested in the1K1C model of acute renal insufficiency. Oral administration ofcinacalcet at 30 mg/kg significantly lowered PTH by approximately 50%for up to 6 hours. This result is consistent with the publishedpreclinical data for cinacalcet (Nemeth et al., J. Pharmacol. Exp.Thor., 308(2):627-35 (2004)) and validates that the 1K1C model of acuterenal insufficiency is an appropriate model for evaluating the activityof calcimimetics for this indication.

The protocol used in this study is as follows. Male Sprague Dawley ratswere purchased from Charles River Laboratories (Hollister, Calif.;requested purchase weight 250-275 g). For studies with test articles,animals were pre-cannulated in the femoral and jugular veins for drugadministration and blood draws, respectively. Animals were maintained ina temperature-controlled environment with a constant 12 hours light/12hours dark cycle and free access to food and water at all times. Allexperimental procedures with animals were performed according to IACUCguidelines.

General anesthesia was induced and maintained by intraperitoneal (IP)injection of sodium pentobarbital (5.2%, 0.4 mL/rat). For animals thatreceived 45 minutes of renal ischemia an additional IP injection ofsodium pentobarbital (5.2%, 0.1 mL/rat) was given to maintain theanesthetic plane. Blood sampling for PTH measurements followingadministration of compounds in normal rats was done under continuousisoflurane anesthesia.

A clean, aseptic technique was used for the entire procedure. After ratswere anesthetized, the abdomen was shaved with electric clippers priorto the operation and the skin cleaned with 70% alcohol solution.

For model development studies, the left femoral vein was cannulated witha PE-10 tube for blood drawing. Both kidneys were exposed via alaparotomy. A right nephrectomy was performed after the right renalpedicle and ureter were ligated with double 2-0 silk sutures. Afterconfirmation of non-bleeding in the right pedicle, the left renal arterywas carefully dissected and clamped with a micro vascular clip to induceleft renal global ischemia. Renal ischemia was confirmed by observationof a global white-grey color change (blanching). The abdominal incisionwas temporarily covered with gauze to help maintain the temperature ofthe abdominal organs. After 45 minutes, the designated period ofischemia, the clip was removed and left renal artery flow was consideredrestored upon observation of a global restoration of red color. Theabdominal incision was closed in layers with 2-0 silk sutures. Theanimal was then recovered from anesthesia. Physiological parametersincluding body temperature (36-37.5° C.) and body weight were measuredthroughout the procedure. Body temperature was monitored and maintainedusing a heat pad rectal probe feedback system.

Approximately 48 hours after the 1K1C surgery (I/R injury), animals weredosed with various compounds to measure effects on plasma PTH andcalcium. In most cases, test articles were administered by IV infusion(infusion time 5, 10 or 30 minutes) although in some studies compoundswere administered by IV bolus or subcutaneous (SC) bolus injection. Fordrug administration and blood draws, animals were anesthetized withisoflurane.

Blood samples were collected periodically throughout the course of thestudy. Serum samples were analyzed for of calcium levels and plasmasamples were analyzed for PTH. Due to the range of baseline PTH valuesfor individual rats, all data are normalized to pre-dosing (baseline)levels. Serum creatinine was measured using a commercially available kitfrom BioAssay Systems (Hayward, Ca), catalog #DICT-500. Analyses wereperformed according to the manufacturer's instructions.

B. Testing Compounds in Renal Insufficiency Model

Compounds with the following sequences were prepared for testing in therenal insufficiency model: Ac-crrrr-NH₂ (SEQ ID NO:4), n=4,Ac-crrrrr-NH₂ (SEQ ID NO:5), n=4, Ac-crrrrrr-NH₂ (SEQ ID NO:6), n=7,Ac-crrrrrrr-NH₂ (SEQ ID NO:7), n=4, and saline control, n=2. Thepeptides were administered to animals at a dose of 3 mg/kg by a30-minute IV infusion. Prior to dosing a blood sample was drawn todetermine baseline, pre-dosing PTH plasma concentration. Results areshown in FIG. 1 as follows: Ac-crrrr-NH₂ (SEQ ID NO:4, diamonds),Ac-crrrrr-NH₂ (SEQ ID NO:5, squares), Ac-crrrrrr-NH₂ (SEQ ID NO:6,triangles), and Ac-crrrrrrr-NH₂ (SEQ ID NO:7, open squares).

Example 2 In Vitro Cell Assay in Hek-293 Expressing the HumanCalcium-Sensing Receptor

Human embryonic kidney (HEK) 293T cells were seeded into a T25 flask at2 million cells per flask and allowed to incubate at 37° C. in 5% CO₂overnight. The day after, these cells were transfected with human CaSRreceptor using lipofectamine 2000 transfection reagent 24 hrs posttransfection, cells were seeded in 384 well plates at 8,000 cells/well.Assays were carried out 48 hours after the transfection. In some cases,EC₅₀ values were determined by measuring inositol monophosphateproduction in the HEK293 cells, stably transfected with the humancalcium-sensing receptor (see Table 1).

The cell culture medium was aspirated from the wells and replaced with28 μL of 1× stimulation buffer (Hepes 10 mM, CaCl2 1 mM, MgCl2 0.5 mM,KCl 4.2 mM, NaCl 146 mM, glucose 5.5 mM, LiCl 50 mM pH 7.4). Cells wereincubated with compounds at various concentrations (1 mM or 300 μM asthe highest and further ½ log serial dilutions) at 37° C. for 1.5 hrsbefore reaction termination. IP₁ production was determined in cellsusing the Cisbio IP-One Tb kit (621PAPEC) and according to manufacturerinstructions. In brief, incubation with the compound was terminated bysequentially adding D2 labeled-IP₁ and cryptate-labeled anti-IP₁ inlysis buffer and further incubating at room temperature for 60 minutes.Plates were read at 620 nm and 668 nm with 314 nm excitation.Non-transfected 293 cells were used as negative control.

The ratio of fluorescence at 668 nm and 620 nm was determined. IP₁concentrations were calculated from standard curves (generated withGraph Pad Prism ver.4) using known concentrations of IP₁ standards.EC₅₀s were calculated based on the values of the fluorescent ratio OD(668 nm)/(OD₆₂₀ nm) using non-linear regression curve fitting in Prismsoftware.

Peptides and conjugates were prepared by solid-phase chemistry at 0.25mmol scale on an ABI automated synthesizer. Sequential coupling ofFmoc-amino acids (4 eq, Anaspec) to Rink-amide resin (NovaBiochem) wasaccomplished using HBTU/DIEA activation. The assembled peptide wascleaved with a TFA cocktail (phenol (5%), triisopropylsilane (2.5%) andwater (2.5%); 10 mL per gram of resin) and isolated by precipitationwith diethyl ether. After purification by C₁₈ HPLC the final product wasisolated in the TFA salt form by lyophilization of appropriatefractions, and characterized by HPLC (>95% purity) and LC-MS (confirmedMW).

Example 3 In Vivo Administration of Compounds with Cationic Subunits

The peptides were administered intravenously at a dose of 0.5 mg/kg intoisoflurane-anesthetized normal Sprague Dawley rats. A control group ofrats was treated with saline. Blood was drawn prior to dosing and everyhour for 4 hours. Rats were maintained under isoflurane anesthesia forthe entire study. The concentration of PTH in the plasma was measured byELISA, detecting the bioactive intact PTH 1-84 (Immutopics Internationalcatalog number 60-2700), and the cumulative area under the curve for AUCwas calculated for the data points inclusive of 1-4 hours. Percent PTHreduction was calculated according to the following formula:AUC_(cmpd treated)/AUC_(saline control)*100.

Example 4 Structure-Activity Relationship Studies In Vivo Activity

The peptides tested identified herein as SEQ ID NO: 26 (Ac-carrrar-NH₂)and as SEQ ID NO:29 (Ac-arrrar-NH₂) were tested in vitro using theHEK293 CaSR transfected cells, according to the procedure in Example 2.The peptides were also tested in vivo, by administering as an IV bolusto normal Sprague Dawley rats at doses of 9 mg/kg for SEQ ID NO:29 andat 0.5 mg/kg for SEQ ID NO: 26. An intravenous (IV) bolus of saline wasused as a control. Plasma (K₂EDTA) PTH levels were assessed prior todosing and 1, 2, 3 and 4 hours after dosing. Rats were maintained underisoflurane anesthesia for the entire study. The results are shown inFIGS. 2A-2B, presented as group average±standard deviation (SD). In FIG.2B, PTH is shown as percent of the baseline pre-dose value.

Example 5 Structure-Activity Relationship Studies D- and L-Amino AcidSubunits

A series of compounds having an L-amino acid residue substituted for aD-amino acid residue were prepared. The compounds were administered asan IV bolus to normal Sprague Dawley rats at a dose of 0.5 mg/kg. Anintravenous (IV) bolus of saline was used as a control. Plasma (K₂EDTA)PTH levels were assessed prior to dosing and 1, 2, 3 and 4 hours afterdosing, and the AUC was calculated as described above. Rats weremaintained under isoflurane anesthesia for the entire study. The resultsare shown in Table 4 above.

Example 6 Structure-Activity Relationship Studies Histamine Release

To evaluate the effect of net positive charge on the histamine releaseassociated with a compound, peptides containing 4 to 7 cationic(arginine) residues were generated and tested for their ability totrigger histamine release in vivo. The tested peptides included (i)Ac-crrrr-NH₂ (SEQ ID NO:4), (ii) Ac-crrrrr-NH₂ (SEQ ID NO:5), (iii)Ac-crrrrrr-NH₂ (SEQ ID NO:6) and (iv) Ac-crrrrrrrr-NH₂; SEQ ID NO:41).

Male Sprague-Dawley rats were obtained (Charles River) pre-cannulated inthe femoral and jugular veins for drug infusion and blood draws,respectively. All IV drug treatments were conducted under anesthetic(isoflurane). Animals were dosed by a 1-minute IV push in a total volumeof 0.5 mL. Blood samples were taken at 5, 15 and 30 minutes following IVbolus to generate plasma (K₂EDTA) samples for histamine analysis. For 30minute IV infusion studies, sample was taken at the end of infusion. Insome case rats in the 1K1C model of acute renal ischemia were used.

An equal volume of saline was injected following each blood draw toreplace lost volume. Approximately 0.2 mL of blood was withdrawn at eachtime point using pre-coated EDTA syringes to facilitate serumcollection.

Histamine ELISAs were performed on diluted plasma using the HistamineEnzyme Immunoassay (EIA) kit (Cat # A05890, SPI-BIO, Montigny leBretonneux, France). The Histamine EIA kit is a derivitization-amplifiedcompetitive enzyme immunoassay which detects histamine within the rangeof 40 μg/mL to 5,500 μg/mL. The samples were analyzed in duplicateaccording to the manufacturer's protocol.

Lyophilized peptides (TFA salts) were weighed and the recorded mass wasadjusted for peptide content. Solutions were prepared by dissolving thematerial in normal saline to generate the desired peptide concentration.In some cases the molarity of peptide was adjusted to allow forinter-peptide comparison. The peptides were administered by IV bolus atan equivalent dose on a per mole basis as SEQ ID NO:41 (i.e., 0.7pmole/rat) by a 1-minute IV bolus and plasma histamine was measuredbefore dosing (pre-dose), 5, 15 and 30 minutes after dosing. Data arepresented as group averages (n=2)±SD. Histamine release is shown as foldchange from pre-dose (baseline) levels. Results are shown in FIG. 3.Data are presented as group averages (n=2)±SD.

Example 7 Structure-Activity Relationship Studies Histamine Release

For in vitro evaluation of histamine release, isolated rat peritonealmast cells were isolated by performing peritoneal lavage using coldHBSS+25 mM HEPES pH 7.4 containing heparin (5 u/mL). Cells were washedtwice in stimulation buffer (HBSS+25 mM HEPES pH 7.4) and incubated with10 μM of compound in stimulation buffer (HBSS+25 mM HEPES pH 7.4) for 15minutes in a 96-well plate (10⁶/well) at 37° C. Cell supernatant wasanalyzed for histamine using histamine EIA kit (Cayman #589651). Data isshown in Table 10.

For the in vivo evaluation of histamine release, compounds were dosed inisoflurane-anesthetized normal rats at 2 mg/kg by IV bolus (administeredover less than one minute). Plasma histamine was measured 5 minutesafter compound administration (Cayman histamine EIA #589651). Data isshown in Table 11.

The abbreviations used herein, and in particular in Tables 10-11 aresummarized here.

Ahx 6-aminohexanoic acid Aib 2-aminoisobutyric acid bAla beta-alanine(3-aminopropionic acid) dHcy D-homocysteine dNle D-norleucine dNvaD-norvaline dPen D-penicillamine EG ethylene glycol spacer,H2N—(CH2CH2—O)4—CH2—CO2H Hcy homocysteine Mpa 3-mercaptopropionic acidor mercaptopropionic acid Nma N-methylalanine PEG poly (ethylene glycol)Sar Sarcosine (N-methylglycine) dHar D-homoarginine GS Glutatione(conjugated) DAP 1,3-diaminopropionic acid

Example 8 Structure-Activity Relationship Studies In Vivo Activity

The compound Ac-c(C)arrrar-NH₂ (SEQ ID NO:3) was prepared for comparisonwith the compound Ac-carrrar-NH₂ (SEQ ID NO:26). In the compoundAc-c(C)arrrar-NH₂ (SEQ ID NO:3) the thiol-containing subunit in positionX₁ is conjugated via a disulfide linkage to an L-Cys residue. The twocompounds were administered via IV bolus to animals with the 1K1C modelof acute renal insufficiency at doses of 0.3 and 0.5 mg/kg. Plasma PTHlevels were assessed prior to dosing and periodically for 24 hours afterdosing. Results are shown in FIG. 10, where data shown are groupaverages±SEM, where as a function of time, in hours, in rats with 1K1Cmodel of acute renal insufficiency, the compound Ac-c(C)arrrar-NH₂ (SEQID NO:3) is represented by squares (0.3 mg/kg, n=5) and * symbols (0.5mg/kg, n=6) and the compound Ac-c(Ac-C)arrrar-NH₂ (SEQ ID NO:141) bytriangles (0.3 mg/kg, n=8) and diamonds (0.5 mg/kg, n=7).

Example 9 Micropore Facilitated Transdermal Delivery of CalcimimeticAgents

To evaluate systemic delivery of a calicimimetic agent, Ac-crrrrrr-NH₂(SEQ ID NO:6) was administered to CD hairless rats transdermally using areservoir. Ac-crrrrrr-NH₂ (SEQ ID NO:6) was applied as a 10% solution insaline to an approximately 1 cm² area on the back of CD® hairless ratsthat were microporated by 5 passes of a 1.0 mm Derma Roller undermoderate pressure. A polystyrene chamber (I.D. 9.5 mm) was glued overthe microporated area of skin to create a drug reservoir in which thesolution of either Ac-crrrrrr-NH₂ (SEQ ID NO: 6) or a saline solutionwere applied. A 10% solution of Ac-crrrrrr-NH₂ (SEQ ID NO: 6) wasadministered in the reservoir chamber on two rats, a saline solutionalone was administered in the reservoir chamber on one rat. Thereservoirs were covered with tape to prevent evaporation. Blood drawswere taken over a 4 hour period and plasma was analyzed for PTH levelsby ELISA. The results are shown in FIG. 11.

Example 10 Sustained Delivery of Calcimimetic Agents by MicroporeFacilitated Transdermal Patch

To further evaluate systemic delivery of a calcimimetic agent,Ac-c(C)arrrar-NH₂ (SEQ ID NO:3) was administered transdermally to normalrats using a transdermal patch. Normal rats were treated withmicroneedle array and transdermal patch system. A small area of fur onthe back of Sprague Dawley rats (˜350 g) was sheared using clippers andan area of skin was microporated using a 14×14 array (˜1 cm²) ofmicroneedles (−0.5 mm). A transdermal patch system containing 10%solution (by weight) of Ac-c(C)arrrar-NH₂ (SEQ ID NO:3) in saline wasplaced over the microporated area and left in place for ˜30 hours. Blooddraws were taken from the rats periodically over the 30 hours and plasmasamples was analyzed for PTH levels by ELISA. The results are shown inFIG. 12.

Example 11 Infusion of Calcimimetic Agents

To further evaluate the PTH lowering effect of the calcimimetic compoundAc-c(C)arrrar-NH₂ (SEQ ID NO:3) was administered by very low dose IVinfusion to normal rats and rats with renal insufficiency to identifythe lowest dose needed to be administered by infusion, transdermal patchsystem or other sustained delivery means to achieve significant PTHreduction. Normal Sprague-Dawley male rats (250-300 g) wereintravenously infused for 6 hours with Ac-c(C)arrrar-NH₂ (SEQ ID NO: 3)at dose rates of 1 μg/kg/hr, 3 μg/kg/hr, and 10 μg/kg/hr. Blood sampleswere taken prior to dosing, at 2 hours, 4 hours, 6 hours (just prior tothe end of infusion; EOI) and 8 hours (2 hrs post EOI) and plasma wasanalyzed for PTH levels by ELISA. The data are shown in FIG. 13, whererats treated with 1 μg/kg/hr (squares), 3 μg/kg/hr (diamonds), and 10μg/kg/hr (triangles) for 6 hours were effective to produce significantreduction in PTH from baseline over the course of infusion.

A similar study was conducted in rats with the 1K1C model of acute renalinsufficiency. 1K1C model rats were intravenously infused withAc-c(C)arrrar-NH₂ (SEQ ID NO: 273 at dose rates of 30 μg/kg/hr and 100μg/kg/hr for 6 hours. Blood samples were taken prior to dosing (Pre), at2 hours, 4 hours, 6 hours (just prior to the end of infusion; EOI), 8hours (2 hrs post EOI) and 24 hours and plasma was analyzed for PTHlevels by ELISA. The data are shown in FIG. 14A (30 μg/kg/hr, diamonds,and 100 μg/kg/hr, squares) and the serum calcium for the animals isshown in FIG. 14B.

1. A compound comprising Ac-c(C)arrrar-NH₂ (SEQ ID NO:3).
 2. Apharmaceutical composition comprising a compound comprisingAc-c(C)arrrar-NH₂ (SEQ ID NO:3) and a pharmaceutically acceptableexcipient.